Methods using persephin and related growth factors

ABSTRACT

A novel growth factor, persephin, which belongs to the GDNF/neurturin family of growth factors, is disclosed. The mouse and rat amino acid sequences have been identified. Mouse and rat persephin genomic DNA sequences have been cloned and sequenced and the respective cDNA sequences identified. In addition, methods for treating degenerative conditions using persephin, methods for detecting persephin gene alterations and methods for detecting and monitoring patient levels of persephin are provided. Methods for identifying additional members of the persephin-neurturin-GDNF family of growth factors are also provided.

This application is a divisional of U.S. patent application Ser. No.08/981,739, filed Aug. 31, 1998 now U.S. Pat. No. 6,232,449, which was acontinuation-in-part of U.S. patent application Ser. No. 08/615,944,filed Mar. 14, 1996 now abandoned.

REFERENCE TO GOVERNMENT GRANT

This invention was made with government support under Grant NumbersNS24679 and CA52524. The government has certain rights in thisinvention.

BACKGROUND OF THE INVENTION

(1) Field of the Invention

This invention relates generally to trophic or growth factors and, moreparticularly, to novel growth factors of the neurturin-GDNF family ofgrowth factors.

(2) Description of the Related Art

The development and maintenance of tissues in complex organisms requiresprecise control over the processes of cell proliferation,differentiation, survival and function. A major mechanism whereby theseprocesses are controlled is through the actions of polypeptides known as“growth factors”. These structurally diverse molecules act throughspecific cell surface receptors to produce these actions.

Growth factors, termed “neurotrophic factors” promote thedifferentiation, growth and survival of neurons and reside in thenervous system or in innervated tissues. Nerve growth factor (NGF) wasthe first neurotrophic factor to be identified and characterized(Levi-Montalcini et al., J. Exp. Zool. 116:321, 1951 which isincorporated by reference). NGF exists as a non-covalently boundhomodimer that promotes the survival and growth of sympathetic, neuralcrest-derived sensory, and basal forebrain cholinergic neurons. Insympathetic neurons this substance produces neurite outgrowth in vitroand increased axonal and dendritic in growth in vivo. (SeeLevi-Montalcini and Booker, Proc. Nat'l Acad Sci 46:384-391, 1960;Johnson et al. Science 210:916-918, 1980; Crowley et al., Cell76:1001-12, 1994 which are incorporated by reference). NGF has effectson cognition and neuronal plasticity, and can promote the survival ofneurons that have suffered damage due to a variety of mechanical,chemical, viral, and immunological insults (Snider and Johnson, AnnNeurol 26:489-506, 1989; Hefti, J. Neurobiol 25:1418-35, 1994 which areincorporated by reference). NGF also is known to extensively interactwith the endocrine system and in immune and inflammatory processes.(Reviewed in Scully and Otten, Cell Biol Int 19:459-469, 1995; Otten andGadient, Int. J. Devl Neurosci 13:147-151, 1995 which are incorporatedby reference). For example, NGF promotes the survival of most cells.(Horigome et al. J Biol Chem 269:2695-2707, 1994 which is incorporatedby reference).

In recent years it has become apparent that growth factors fall intoclasses, i.e. families or superfamilies based upon the similarities intheir amino acid sequences. These families include, for example, thefibroblast growth factor family, the neurotrophin family and thetransforming growth factor-beta (TGF-β) family. As an example of familymember sequence similarities, TGF-β family members have 7 canonicalframework cysteine residues which identify members of this superfamily.

NGF is the prototype of such a family of growth factors. Brain-derivedneurotrophic factor (BDNF), the second member of this family to bediscovered, was shown to be related to NGF by virtue of the conservationof all six cysteines that form the three internal disulfides of the NGFmonomer (Barde, Prog Growth Factor Res 2:237-248, 1990 and Liebrock etal. Nature 341:149-152, 1989 which are incorporated by reference). Byutilizing the information provided by BDNF of the highly conservedportions of two factors, additional members (NT-3, NT-4/5) of thisneurotrophin family were rapidly found by several groups (Klein, FASEB J8:738-44, 1994 which is incorporated by reference).

Neurotrophic factors structurally unrelated to NGF have been recentlyidentified. These include factors originally isolated based upon a“neurotrophic action” such as ciliary neurotrophic factor (CNTF) (Lin etal., Science 246:1023-5, 1989 which is incorporated by reference) alongwith others originally isolated as a result of non-neuronal activities(e.g. fibroblast growth factors (Cheng and Mattson Neuron 1:1031-41,1991 which is incorporated by reference), IGF-I (Kanje et al, Brain Res486:396-398, 1989 which is incorporated by reference) leukemiainhibitory factor (Kotzbauer et al, Neuron 12:763-773, 1994 which isincorporated by reference).

Glial-derived neurotrophic factor (GDNF), is one such neurotrophicfactor structurally unrelated to NGF. GDNF was, thus, a unique factor,which, up until now, was not known to be a member of any subfamily offactors. The discovery, purification and cloning of GDNF resulted from asearch for factors crucial to the survival of midbrain dopaminergicneurons, which degenerate in Parkinson's disease. GDNF was purified fromrat B49 glial cell conditioned media (Lin et al., Science 260:1130-2,1993 which is incorporated by reference). Sequence analysis revealed itto be a distant member of the TGF-β superfamily of growth factors,having approximately 20% identity based primarily on the characteristicalignment of the 7 canonical framework cysteine residues (Lin et al.,Science 260:1130-2, 1993 which is incorporated by reference). Thus, GDNFcould possibly have represented a new subfamily within the TGF-βsuperfamily.

Recombinant GDNF produced in bacteria specifically promotes the survivaland morphological differentiation of dopaminergic neurons (Lin et al.,Science 260:1130-2, 1993); Tomac et al., Nature 373:335-9, 1995; Beck etal., Nature 373:339-41, 1995 and Ebendal et al., J Neurosci Res40:276-84, 1995 which are incorporated by reference) and motor neurons(Henderson et al., Science 266:1062-4, 1994; Yan et al., Nature373:341-4, 1995; and Oppenheim et al., Nature 373:344-6, 1995 which areincorporated by reference). Overall, GDNF was a more potent factor forpromoting the survival of motor neurons than the other factors, and itwas the only factor that prevented neuronal atrophy in response to theselesions, thereby positioning it as a promising therapeutic agent formotor neuron diseases.

It is now generally believed that neurotrophic factors regulate manyaspects of neuronal function, including survival and development infetal life, and structural integrity and plasticity in adulthood. Sinceboth acute nervous system injuries as well as chronic neurodegenerativediseases are characterized by structural damage and, possibly, bydisease-induced apoptosis, it is likely that neurotrophic factors playsome role in these afflictions. Indeed, a considerable body of evidencesuggests that neurotrophic factors may be valuable therapeutic agentsfor treatment of these neurodegenerative conditions, which are perhapsthe most socially and economically destructive diseases now afflictingour society. Nevertheless, because different neurotrophic factors canpotentially act preferentially through different receptors and ondifferent neuronal or non-neuronal cell types, there remains acontinuing need for the identification of new members of neurotrophicfactor families for use in the diagnosis and treatment of a variety ofacute and chronic diseases of the nervous system.

SUMMARY OF THE INVENTION

Briefly, therefore, the present invention is directed to theidentification and isolation of substantially purified factors thatpromote the survival and growth of neurons as well as non-neuronalcells. Accordingly, the inventors herein have succeeded in discoveringnovel protein growth factors belonging to a family of growth factors forwhich GDNF was the first known member. The first such newly discoveredfamily member was neurturin and this is the subject of application Ser.No. 08/519,777, now U.S. Pat. No. 5,739,307. Based upon the sequence ofGDNF and neurturin the inventors herein have discovered another memberof the GDNF-Neurturin family of growth factors referenced herein aspersephin (PSP). This growth factor is believed to show at least 85%sequence identity among homologous sequences from different mammalianspecies although sequence homology may be as low as 65% in non-mammalianspecies such as avian species. Persephin proteins identified hereininclude mouse sequences as set forth in SEQ ID NOS:79, 80 and 81 (FIG.11; amino acid residues 52 through 140, 47 through 142, and 9 through142, respectively) and rat sequences as set forth in SEQ ID NOS:82 and83 (FIG. 14; amino acid residues 1 through 89 and 1 through 91,respectively). In addition, human persephin is identified by virtue ofits having at least 85% sequence homology with its ortholog, maturemouse persephin, along with the identification of certain conservedamino acid residues contained within human persephin as shown in FIG.15. Thus, it is believed that human persephin will have 28 amino acidsin the aligned sequence between the first and seventh canonicalframework cysteine residues as set forth in FIG. 15 with residuesnumbered from the N-terminal end of the family member aligned sequencebeing (1) Cys, (3) Leu, (10) Val, (13) Leu, (14) Gly, (15) Leu, (16)Gly, (17) Tyr, (21) Glu, (25) Phe, (26) Arg, (27) Tyr, (28) Cys, (30)Gly, (32) Cys, (44) Leu, (47) Leu, (58) Cys, (59) Cys (61) Pro, (66)Asp, (69) Phe, (70) Leu, (71) Asp, (83) Ser, (84) Ala, (87) Cys, and(89) Cys.

Persephin has been identified and obtained by a method based upon theconserved regions of the GDNF-Neurturin family discovered by theinventors herein. Accordingly, a new method has been devised thatutilizes degenerate primers constructed from the sequences of theseconserved regions for use in the polymerase chain reaction procedure. Byutilizing this method the mouse and rat orthologs of the new familymember, persephin, have been identified and obtained.

The present invention thus provides both amino acid sequences andnucleotide sequences that encode mouse and rat persephin as set forth inthe amino acid sequences of SEQ ID NO:79-83 and nucleotide sequences ofSEQ ID NOS:84 and 85. Because of the close homology between the mouseand rat sequences (95% sequence identity), it is believed that the humanpersephin sequence will show a high sequence homology to the mouse andrat sequences.

Expression vectors and stably transformed cells are also within thescope of this invention. The transformed cells can be used in a methodfor producing persephin.

In another embodiment, the present invention provides a method forpreventing or treating neuronal degeneration comprising administering toa patient in need thereof a therapeutically effective amount ofpersephin. A patient may also be treated by implanting transformed cellswhich express persephin or a DNA sequence which encodes persephin into apatient, or cells cultured and expanded by growth in persephin.

The present invention also provides compositions and methods fordetecting persephin. One method is based upon persephin antibodies andother methods are based upon detecting mRNA and cDNA or genomic DNAencoding persephin using recombinant DNA techniques.

Among the several advantages found to be achieved by the presentinvention, therefore, may be noted the provision of a new growth factor,persephin, for use in preventing the atrophy, degeneration or death ofcertain cells, in particular neurons; the provision of human persephinby making available the specific sequences of murine and rat persephinfrom which the human sequence can be identified and obtained; theprovision of other members of the neurturin-persephin-GDNF family ofgrowth factors by making available new methods capable of obtainingother family members; the provision of methods for obtaining persephinby recombinant techniques; the provision of methods for preventing ortreating diseases producing cellular degeneration and, particularlyneuronal degeneration; the provision of methods that can detect andmonitor persephin levels in a patient; and the provision of methods thatcan detect alterations in the persephin gene.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the purification scheme for preparing neurturin fromCHO cells;

FIGS. 2A-B illustrates the characterization of fractions eluted fromMono S column in purifying neurturin showing (a) electrophoresis of eachfraction on a SDS-polyacrylamide gel and visualization of the proteinsby silver stain and (b) the neurotrophic activity present in eachfraction in the superior cervical ganglion survival assay;

FIGS. 3A-C illustrates the ability of neurturin to maintain survival ofsuperior cervical ganglionic cells in culture showing (a) positivecontrol cells maintained with nerve growth factor (NGF) (b) negativecontrol cells treated with anti-NGF antibodies showing diminishedsurvival and (c) cells treated with anti-NGF and neurturin(approximately 3 ng/ml) showing survival of neurons;

FIG. 4 illustrates the concentration-response effect of neurturin in thesuperior cervical ganglion survival assay;

FIG. 5 illustrates the homology of the amino acid sequences for themature growth factors, human neurturin (hNTN), mouse neurturin (mNTN),rat GDNF (rGDNF), mouse GDNF (mGDNF) and human GDNF (hGDNF) withidentical amino acid residues enclosed in boxes;

FIG. 6 illustrates the tissue distribution of neurturin mRNA and themRNA for GDNF using RT/PCR analysis on RNA samples obtained fromembryonic day 21 (E21) and adult rats;

FIG. 7 illustrates the cDNA (SEQ ID NO:11) and encoded amino acidsequence (SEQ ID NO:7) of human pre-pro neurturin [(SEQ ID NO:11)]showing the pre- region from nucleic acid 1 through 57 (SEQ ID NO:17),the pro- region from nucleic acid 58 through 285 (SEQ ID NO:20) , humanneurturin from nucleic acid 286 through 591 (SEQ ID NO:9) and the splicesite between nucleic acids 169 and 170 which defines the coding sequenceportion of two exons from nucleic acids 1 through 169 (SEQ ID NO:27) and170 through 594 (SEQ ID NO:28);

FIG. 8 illustrates the cDNA (SEQ ID NO:12) and encoded amino acidsequence (SEQ ID NO:8) of mouse pre-pro neurturin [(SEQ ID NO:12)]showing the pre-region from nucleic acid 1 through 57 (SEQ ID NO:18),the pro- region from nucleic acid 58 through 285 (SEQ ID NO:21), mouseneurturin from nucleic acid 286 through 585 (SEQ ID NO:10) and thesplice site between nucleic acids 169 and 170 which defines the codingsequence portion of two exons from nucleic acids 1 through 169 (SEQ IDNO:29) and 170 through 588 (SEQ ID NO:30);

FIG. 9 illustrates the mouse cDNA sequence containing a 5′ non-codingregion (SEQ ID NO:13) and a 3′ non-coding region (SEQ ID NO:14) each ofwhich are contiguous to the coding region of pre-pro neurturin;

FIG. 10 illustrates the percent neuronal survival in E18 rat nodoseganglia neurons treated 24 hours post-plating for NTN, GDNF, BDNF, NGFand AMO;

FIG. 11 illustrates the nucleotide (SEQ ID NO:105) and amino acidsequence (SEQ ID NO:111) of murine persephin (SEQ ID NO:79, 80 and 81;amino acid residues 52 through 140, 47 through 142, and 9 through 142,respectively);

FIG. 12 illustrates the family member sequence identity in the regionbetween the first and seventh canonical framework cysteine residuesaligned beginning with the first canonical framework cysteine for murineGDNF (SEQ ID NO:87), murine neurturin (NTN) (SEQ ID NO:88) and murinepersephin (PSP) (SEQ ID NO:89);

FIG. 13 illustrates the partial sequence of rat persephin cDNA (SEQ IDNO:97) and the corresponding amino acid sequence (aa19-aa91 of SEQ IDNO:83) obtained by the technique of rapid amplification of cDNA ends;

FIG. 14 illustrates the partial sequence beginning with the firstcanonical framework cysteine for rat persephin (SEQ ID NO:83) and thecorresponding polynucleotide sequence (SEQ ID NO:86);

FIG. 15 shows the family member aligned partial amino acid sequencesfrom the first through the seventh canonical framework cysteine residuesillustrating family member sequence homology of the mature growthfactors, human GDNF (aa41-aa133 of SEQ ID NO:76), rat GDNF (aa41-aa133of SEQ ID NO:78), mouse GDNF (aa41-aa133 of SEQ ID NO:77), humanneurturin (NTN) (SEQ ID NO:31), mouse neurturin (SEQ ID NO:32), ratpersephin (PSP) (SEQ ID NO:82) and mouse persephin (SEQ ID NO:79) inwhich boxes enclose the 28 conserved amino acid residues present in all;

FIG. 16 illustrates the sequences of TGF-β superfamily members alignedusing the Clustal method, from the first canonical framework cysteine tothe end of the sequence for transforming growth factor-β1 (TGFβ1) SEQ IDNO:150), transforming growth factor-β2 (TGFβ2) (SEQ ID NO:151),transforming growth factorβ3 (TGFβ3) (SEQ ID NO:152), inhibin β A(INHβA) (SEQ ID NO:153), inhibin β B (INHβB) (SEQ ID NO:154), the nodalgene (NODAL) (SEQ ID NO:155), bone morphogenetic proteins 2 and 4 (BMP2and BMP4) (SEQ ID NOs:156 and 157, respectively), the Drosophiladecapentaplegic gene (dpp) (SEQ ID NO:158), bone morphogenetic proteins5-8 (BMP5, BMP6, BMP7 and BMP8) (SEQ ID Nos:159, 160, 161 and 162,respectively), the Drosophila 60A gene family (60A) (SEQ ID NO:163),bone morphogenetic protein 3 (BMP3) (SEQ ID NO:164), the Vg1 gene (SEQID NO:165), growth differentiation factors 1 and 3 (GDF1 and GDF3) (SEQID Nos:165 and 167, respectively), dorsalin (dsrin) SEQ ID NO:168),inhibin α (INHα) (SEQ ID NO:169), the MIS gene (MIS) (SEQ ID NO:170),growth factor 9 (GDF-9) (SEQ ID NO:171), gial-derived neurotrophicgrowth factor (GDNF) (SEQ ID NO:172), and neurturin (NTN) (SEQ IDNO:173);

FIG. 17 illustrates full length murine persephin gene (SEQ ID NO:131),the amino acid sequence which includes at least a portion of the pre-proregion encoded by the nucleotide sequence in the first reading framefrom the initiator methionine codon through the stop codon at nucleotidepositions 244-246 (SEQ ID NO:132) and the amino acid sequence whichincludes mature persephin in the second reading frame from nucleotideposition 2 through the stop codon at positions 557-559 (SEQ ID NO:133);

FIG. 18 illustrates full length rat persephin gene (SEQ ID NO:134), theamino acid sequence which includes at least a portion of the pre-proregion encoded by the nucleotide sequence in the first reading framefrom the initiator methionine codon through the stop codon at nucleotidepositions 244-246 (SEQ ID NO:135) and the amino acid sequence whichincludes mature persephin in the second reading frame from nucleotideposition 2 through the stop codon at positions 557-559 (SEQ ID NO:136);

FIG. 19 illustrates a western blot analysis using anti-persephinantibodies to detect persephin protein in cell lysates from COS monkeycells transfected with the murine persephin gene (lane 2) or the ratpersephin gene (lane 3) compared to cells transfected with thenon-recombinant vector alone (pCB6, lane 4) and the mature proteinproduced by E. Coli (lane 1);

FIGS. 20A-B illustrates the murine chimeric molecules (A) PSP/NTN (SEQID NO:141) containing the persephin fragment (residues 1-63) and theneurturin fragment (residues 68-100) and (B) NTN/PSP (SEQ ID NO:146)containing the neurturin fragment (residues 1-67) and the persephinfragment (residues 64-96) with the arrow indicating the crossover pointin each;

FIGS. 21A-B illustrates the survival promoting effect of persephin inmurine embrionic day-14 mesencephalic cells cultured for three days (a)in the absence of persephin where almost all of the cells are dead and(b) in the presence of persephin (100 ng/ml) where substantial neuronalcall survival is evident; and

FIG. 22 illustrates RT/PCT survey for persephin expression in adultmouse tissues showing persephin expression by Kidney cells.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is based upon the identification, isolation andsequencing of a DNA molecule that encodes a new growth factor,persephin. Because of the sequence similarity to neurturin and GDNF,persephin is believed to be capable of promoting cell survival and, inparticular, the survival of neurons. Prior to this invention, persephinwas unknown and had not been identified as a discrete biologicalsubstance nor had it been isolated in pure form.

The growth factor, neurturin (NTN) was identified and isolated as setforth in application Ser. No. 08/519,777 filed Aug. 28, 1995, now U.S.Pat. No. 5,739,307, which is incorporated in its entirety by reference.From the sequence of neurturin and the sequence of the closely relatedgrowth factor, glial-derived neurotrophic factor (GDNF), the inventorsherein have devised and pursued strategies to find additional relatedfactors. Neurturin is approximately 40% identical to GDNF, but less than20% identical to any other member of the TGF-β superfamily. Togetherthese two proteins define a new subfamily within the TGF-β superfamily.Several sequence regions within neurturin and GDNF were identified thatare highly conserved, such that they are likely to be present in anyadditional members of this subfamily. This sequence information cantherefore be used to isolate previously unknown members of thissubfamily by designing degenerate oligonucleotides to be used as eitherprimers in PCR reactions or as probes in hybridization studies.

Using the new degenerate primer PCR strategy described in Example 11 ofapplication Ser. No. 08/519,777, now U.S. Pat. No. 5,739,307, theinventors herein have succeeded in identifying a third factor,persephin, that is approximately 40-50% identical to both GDNF andneurturin. Primers corresponding to the amino acid sequence fromconserved regions of neurturin and GDNF (SEQ ID NO:42 and SEQ ID NO:44)were used to amplify a 77 nt fragment from rat genomic DNA. Theresulting products were subcloned into the Bluescript KS plasmid andsequenced. The sequence of one of the amplified products predicted aminoacid sequence data internal to the PCR primers that was different fromthat of GDNF or neurturin but had more than 20% identity with GDNF andneurturin, whereas the sequences of other amplified products we obtainedcorresponded to GDNF or neurturin, as would be expected. The 22nucleotide sequences (SEQ ID NO:90) was then aligned with the ratsequences of GDNF and neurturin and found to be unique. This novelsequence, thus, suggested that we had identified a new family memberreferenced herein as persephin.

To obtain additional persephin sequence information, primers containingthe unique 22 nucleotide sequence of the amplified fragment were used inthe rapid amplification of cDNA ends (RACE) technique (Frohman, M. A.Methods in Enzymology 218:340-356, 1993) using cDNA obtained fromneonatal rat brain. An approximately 350 nt fragment was obtained fromthis PCR reaction which constituted a partial rat persephin cDNAsequence of approximately 350 nucleotides (SEQ ID NO:106). The predictedamino acid sequence of this cDNA was compared to that of GDNF andneurturin, and found to have approximately 40% identity with each ofthese proteins. Importantly, the characteristic spacing of the canonicalframework cysteine residues in members of the TGF-β superfamily waspresent. Furthermore, in addition to the region of similarity encoded bythe degenerate primers used to isolate persephin, another region of highhomology shared between GDNF and neurturin, but absent in other membersof the TGF-β superfamily, was also present in persephin

GDNF ACCRPVAFDDDLSFLDD (aa 60-76) (SEQ ID NO:98)

NTN PCCRPTAYEDEVSFKDV (aa 61-77) (SEQ ID NO:99)

PSP PCCQPTSYAD-VTGLDD (aa 57-72) (SEQ ID NO:100)

(Amino acid numbering uses the first Cys residue as amino acid 1).

With the confirmation that persephin was indeed a new member of theGDNF/NTN subfamily, we isolated murine genomic clones of persephin toobtain additional sequence information. Primers corresponding to ratcDNA sequence were used in a PCR reaction to amplify a 155 nucleotide(nt) fragment from mouse genomic DNA which was homologous to the ratpersephin cDNA sequence. These primers were then used to obtain murinepersephin genomic clones from a mouse 129/Sv library in a P1bacteriophage vector (library screening service of Genome Systems, Inc.,St. Louis, Mo.).

Restriction fragments (3.4 kb Nco I and a 3.3 kb Bam H1) from this P1clone containing the persephin gene were identified by hybridizationwith a 210 nt fragment of persephin obtained by PCR using mouse genomicDNA and persephin-specific primers. The Nco I and Bam H1fragments weresequenced and found to encode a stretch of amino acids corresponding tothat present in the rat persephin RACE product, as well as beinghomologous to the mature regions of both neurturin and GDNF (FIG. 11).

When the amino acid sequences of mature murine GDNF, NTN and PSP arealigned using the first canonical framework cysteine as the startingpoint, which is done because alternations in the cleavage sites betweenfamily members creates variability in the segments upstream of the firstcysteine, persephin (91 amino acids) is somewhat smaller than eitherneurturin (95 amino acids) or GDNF (94 amino acids). The overallidentity within this region is about 50% with neurturin and about 40%with GDNF (FIG. 12).

Further nucleotide sequencing of the murine persephin NcoI fragmentrevealed the nucleotide sequence of the entire murine persephin gene asshown in FIG. 17. In addition, the entire rat persephin gene has beendetermined by sequencing a PCR amplified fragment of rat genomic DNA asshown in FIG. 18. In both the murine and rat persephin gene, an openreading frame extends from the sequence coding for an initiatormethionine up to a stop codon at positions 244-246. However, somewherein this sequence an apparent anomaly occurs such that the sequenceencoding the RXXR cleavage site (positions 257-268) and the sequencecorresponding to the mature persephin protein (positions 269-556) arenot co-linear with this open reading frame. Instead, a second readingframe encodes the cleavage site and the mature persephin. The two cogentreading frames are shown in FIGS. 17 and 18. Irrespective of thisapparent anomaly, mammalian cells express persephin from either themurine or rat full length genomic sequence (see Example 14 below).

The N-terminus of persephin was predicted by reference to the N-terminalregions of neurturin or GDNF. Using neurturin sequence homology andcleavage signals, a characteristic RXXR cleavage motif is presentbeginning 9 residues upstream of the first canonical framework cysteineof persephin which would suggest that mature murine persephin wouldcontain 5 amino acids (ALAGS) (SEQ ID NO:103) upstream of this cysteine(as does neurturin). The corresponding 5 amino acids in rat persephinare ALPGL (SEQ ID NO:112). Using these parameters, mature persephinwould consist of 96 amino acids and have a predicted molecular mass of10.4 kD. Using GDNF sequence homology and cleavage signals, on the otherhand suggests that the N-terminus upstream from the first cysteine ofpersephin could be longer, in accord with that observed for GDNF whichis 40 residues. A characteristic RXXR cleavage motif is thus located 47residues upstream of the first cysteine and this would suggest thatmature persephin would contain 43 amino acids(VRIPGGLPTPQFLLSKPSLCLTILLYLALGNNHVRLPRALAGS) (SEQ ID NO:104) upstreamof this cysteine. Using these parameters, mature persephin would consistof 134 amino acids and have a predicted molecular mass of 14.5 kD. Thus,mature persephin may exist in either or both of the 96 amino acidpredicted 10.4 kD or for the 134 amino acid predicted 14.5 kD form.

By “mature” growth factor reference is made to the secreted form of thegrowth factor in which any pre- or pro- regions have been cleaved andwhich may exist as a monomer or, by analogy to other members of theTGF-β superfamily, in the form of a homodimer linked by disulfide bonds.

The discovery of the new growth factor, persephin, as described above isa result of the prior discovery by the inventors herein of neurturin.Thus, the experiments leading to the discovery of neurturin are relevantto the current discovery of persephin and to the predicted human form ofpersephin as well as to the biological activity of persephin.

Neurturin was identified and isolated by the inventors herein fromconditioned medium for CHO cells. The initial neuronal survivalpromoting activity was identified by the inventors in a partiallypurified preparation of this CHO-conditioned medium. Preparation ofconditioned medium for a given cell line is well known in the art (forexample, see Reid, in Methods in Enzymology Vol. LVIII, Cell Culture,Jakoby and Pastan, Eds., Academic Press, San Diego, pp 161-164, 1979;Freshney, Culture of Animal Cells in A Manual of Basic Technique, 2dEd., Wiley-Liss, NY, p. 84, 1987 which are incorporated by reference).Thus, although in the present work CHO cells were cultured and theconditioned medium used to identify and to obtain neurturin in purifiedform, one skilled in the art will readily appreciate that any cell thatexpresses neurturin can be used as a source. Some of the cells thatexpress neurturin are identified below in Example 9 and the inventorsherein believe that any of the cells identified as expressing neurturincan be used to obtain conditioned medium from which neurturin can beisolated.

In the isolation of neurturin from the CHO cell conditioned medium, aninitial crude conditioned medium can be obtained by centrifugationand/or filtration to remove cellular debris. For further purification,one skilled in the art will readily appreciate that any of a number ofmethods known in the art can be used to isolate and purify neurturinfrom a biological sample such as affinity chromatography, ion exchangechromatography, preparative electrophoresis or the like wherein themethods are used either individually or in combination.

The cell survival promoting effect of neurturin can be assessed in anysuitable system for assessing cell survival. The inventors hereinbelieve that neurturin can promote survival in a variety of differenttissues based upon what is known for other growth factors and upon theobservation that neurturin is expressed in a number of tissues in whichit is believed to have a survival promoting effect.

By virtue of the degree of sequence identity of persephin with itsparalogs, neurturin and GDNF and the known actions of these substancesin promoting the survival and growth of neuronal and non-neuronaltissues, it is also believed that persephin will promote survival andgrowth in neuronal tissues as well as a variety of non-neuronal tissues.Indeed, the inventors herein have identified brain, kidney, and hearttissues as tissues expressing persephin which further supports theconclusion that persephin can act to promote growth and survival inneuronal and non-neuronal cells.

In the work reported herein, neuronal activity for neurturin wasassessed using a sympathetic neuronal survival assay (sympatheticcervical ganglia, SCG) which has been extensively characterized (Martinet al, J Cell Biol 106:829-844, 1989; Deckwerth and Johnson, J Cell Biol123:1207-1222, 1993; which are incorporated by reference) (see FIG. 3).We also show the survival promoting effects of neurturin on sensoryneurons (See FIG. 10). In the same sympathetic and sensory neuronal cellassays, persephin showed little survival potentiating activity. However,in a preparation of CNS neuronal cells of mesencephalic origin,persephin showed neuronal survival potentiating activity. This suggeststhat persephin will be applicable in the treatment or prevention ofdiseases involving neuronal degeneration in the CNS such as, forexample, Parkinson's disease.

By way of illustrating the methods used in the above survival assays,the SCG assay involved the culturing of cells obtained from superiorcervical ganglia of rat embryo for 5 days at 37° C. in medium containingnerve growth factor (NGF). The medium was then exchanged with a mediumcontaining no NGF and containing anti-NGF antiserum. Removal of NGFresults normally in death of the neurons in 24-72 hours. Neuronalsurvival was visually assessed under a microscope on days 7-8. Maximumneuronal survival criteria included lack of degeneration of bothneuronal cell bodies and neurites. Cell body degeneration was indicatedwhen the neuronal cell body was reduced in size, showed irregularmembrane swellings, contained vacuoles, or had lost refractility. Afield of neurites was scored as showing signs of disintegration whenswellings and blebs appeared along the neurite bundles. Survival wasdetermined by comparison with neurons grown in the presence of NGF(positive control) or in the absence of NGF with NGF antisera (negativecontrol).

Activity was quantitated by calculation of a “survival unit”. The totalsurvival units in a sample were defined as the minimal volume of analiquot of the sample which produced maximal survival divided into thetotal volume of that sample. For example, a volume of 600 ml was elutedfrom the heparin agarose column and from this eluate, 12.5 μl was theminimum volume that promoted maximal volume. Thus, the survival units inthe eluate from the heparin agarose column was 48,000. Specific activitywas calculated as the survival units divided by the mg total protein.The intrinsic activity of neurturin is expressed herein in concentrationunits of pg/ml or pM promoting maximal or half-maximal survival. Asshown in FIG. 5, a concentration-response curve of purified neurturinprotein indicates that the intrinsic activity of neurturin expressed asan EC₅₀ is approximately 1.5 ng/ml or approximately 50 pM and an EC₁₀₀is approximately 3ng/ml or approximately 100 pM.

Survival units were determined in an assay using approximately 1200neurons in a 0.5 ml culture assay and a culture period of 48 hoursfollowing addition of the fraction. Survival was assessed visually afterthe 48 hours. Intrinsic activity as shown in FIG. 4 was determined in anassay using approximately 2700 neurons and a culture period of 72 hours.Survival was assessed by fixing the neurons and counting the number ofsurviving neurons. Because the stability, as assessed by half-life ofactivity, for neurturin decreases as the number of neurons increases,the intrinsic activity measurement would be expected to be lower thanthat predicted by Specific Activity determinations. The intrinsicactivity measurement would also be expected to be lower than thatpredicted by specific activity because the survival was measured after72 hours instead of 48 hours.

The purification of neurturin is described in detail in Example 1 below.The conditioned medium starting material was prepared from a derivativeof DG44 Chinese hamster ovary cells, DG44CHO-pHSP-NGFI-B (Day et al, JBiol Chem 265:15253-15260, 1990 which is incorporated by reference). Theinventors herein have also isolated neurturin in partially purified formfrom conditioned medium of other derivatives of DG44 Chinese hamsterovary cells and these other cells could be used equally as well as theDG44CHO-pHSP-NGFI-B cells as could the parent DG44 Chinese hamster ovaryCells, ovary cells from other species and cells from other tissues suchas those known to express neurturin (see example 9). In preparing theconditioned medium, cells were placed in serum free medium for 2 days atwhich time conditioned medium is collected and the medium replenished.This cycle was repeated to yield 5 harvests of conditioned medium fromeach batch of CHO cells. The collected media was centrifuged to removecellular debris.

The first step in purification of neurturin from the CHO cellconditioned medium involved the introduction of the conditioned mediumonto a heparin agarose column and the elution of partially purifiedneurturin therefrom. This step resulted in an 111 fold increase in thespecific activity and purification of the protein. The buffer used toapply the medium to the column contains 0.5 M NaCl. At thisconcentration of NaCl the neurturin binds to the heparin agarose matrix.The inventors herein believe that based upon their isoelectric points,LIF and CNTF would either not bind to the heparin agarose matrix or bewashed away from the matrix with buffer containing 0.5 M NaCl. Thus,this step would be expected to isolate neurturin from growth factorssuch as LIF and CNTF. After washing the column, neurturin was elutedfrom the column using 1.0 M NaCl.

Fur further purification, the eluted material was then diluted andintroduced into a column containing SP SEPHAROSE® High Performance ionexchange resin (Pharmacia, Piscataway, N.J.). Material eluted from thiscolumn was further purified using fast protein liquid chromatography(FPLC) on a Chelating Superose HR 10/2 column charged with Cu**(Pharmacia, Piscataway, N.J.). Eluted fractions from the Cu** superosecolumn were introduced into a Mono S HR 5/5 cation exchange column(Pharmacia, Piscataway, N.J.) for further FPLC purification. Thecomposition of the proteins in the Mono S fractions were analyzed usingnon-reducing SDS-PAGE and silver staining.

Fractions collected from the columns at each stage of purification wereassayed for biological activity using the neuronal survival assay andfor protein content using the dye binding method of Bradford (AnalBiochem 72:248-254, 1976 which is incorporated by reference) with aBio-Rad protein assay dye reagent (Bio-Rad Laboratories, Inc., Hercules,Calif.). The progressive purification using the above steps is shown intable 1.

TABLE 1 Specific Protein^(a) Activity^(b) Activity^(d) YieldPurification (mg) (units) (units/mg) (%) (fold) Conditioned 5000 48000^(c) 9.6 — — Medium Heparin 45 48000 1068 100   111 Agarose SPSepharose 5.3 48000 9058 100   943 Cu++ 0.31 30000 96700  62  10070Superose Mono S 0.004 15000 3750000  31 390000 ^(a)mg protein wasdetermined using the dye binding method of Bradford (Anal Biochem72:248, 1976). ^(b)The total activity units or survival units in asample were defined as the minimal volume of an aliquot of the samplewhich produced maximal survival divided into the total volume of thatsample. ^(c)Activity for Conditioned Medium was derived from theassumption that 100% of the activity was recovered in the heparinagarose fraction because the activity of conditioned medium was too lowto be directly assayed. ^(d)Specific Activity was the Activity unitsdivided by the mg total protein.

The results of this analysis along with the results of the neuronalsurvival assay of fractions revealed that a protein having an apparentmolecular weight of about 25 kD co-purified with the sympathetic neuronsurvival activity.

The purified material isolated from CHO cell conditioned medium was usedto determine partial amino acid sequences of the protein in CHO cellconditioned medium and subsequently as a basis for determining thesequences in different species. The N-terminal amino acid sequence wasdetermined using an automated protein/peptide sequencer and the first 16amino acids were considered to be, with uncertainty as to position 6,Ser-Gly-Ala-Arg-Pro-Xaa-Gly-Leu-Arg-Glu-Leu-Glu-Val-Ser-Val-Ser whereXaa was an unknown amino acid (SEQ ID NO:3). Internal amino acidfragments were obtained from the purified material following digestionwith protease enzymes and the sequences determined. Three internalfragments thus obtained were (1) with uncertainty as to positions 1, 2and 6, Xaa₁-Cys-Ala-Gly-Ala-Xaa₂-Glu-Ala-Ala-Val where Xaa₁ was unknownamino acid, Xaa₂ was Ser or Cys (SEQ ID NO:4); (2) with uncertainty asto positions 1, 2, 4, 10, 17 and 22,Xaa₁-Xaa₂-Val-Glu-Ala-Lys-Pro-Cys-Cys-Gly-Pro-Thr-Ala-Tyr-Glu-Asp-Xaa₃-Val-Ser-Phe-Leu-Ser-Valwhere Xaa₁ and Xaa₂ were unknown, Xaa₃ was Gln or Glu (SEQ ID NO:5) and(3) Try-His-Thr-Leu-Gln-Glu-Leu-Ser-Ala-Arg (SEQ ID NO:6). Based uponthese partial amino acid sequences, DNA probes and primers can be madeand used to obtain cDNA clones from different species based upon highsequence conservation between mammalian species. The human cDNA andinferred amino acid sequence is shown in FIG. 7 and the mouse cDNA andinferred amino acid sequence is shown in FIG. 8.

The cDNA clone from mouse was 1.0 kb having an open reading frame of 585nucleotides (SEQ ID NO:12) encoding the mouse pre-pro neurturin protein(SEQ ID NO:8, FIG. 8). In addition, non-coding regions have beenidentified at both the 5′ and 3′ ends of the coding region as shown inFIG. 9. (SEQ ID NO:13, 5′ non-coding region, nucleic acids −348 through−1; SEQ ID NO:14, 3′ non-coding region, nucleic acids 589 through 675).The mouse neurturin sequence can be used to obtain PCR primers for usein identifying homologs from other species. A human 192 nucleotidefragment from human genomic DNA was amplified by this method and furtherused to screen a human genomic library to obtain clones containing thehuman neurturin genomic locus. The human cDNA sequence was deduced fromthe sequencing of these clones. (FIG. 7, cDNA sequence of human pre-proneurturin).

Reference to persephin or to neurturin herein is intended to beconstrued to include growth factors of any origin which aresubstantially homologous to and which are biologically equivalent,respectively, to the persephin characterized and described herein or tothe neurturin characterized and described herein. Such substantiallyhomologous growth factors may be native to any tissue or species and,similarly, biological activity can be characterized in any of a numberof biological assay system. Reference to pre-pro neurturin herein isintended to be construed to include pre-pro growth factors containing apre- or leader or signal sequence region, a pro- sequence region andneurturin as defined herein.

The terms “biologically equivalent” are intended to mean that thecompositions of the present invention are capable of demonstrating someor all of the same growth properties in a similar fashion, notnecessarily to the same degree as the neurturin isolated from the CHOcell conditioned medium herein or recombinantly produced human or mouseor rat neurturin or persephin as the case may be.

By “substantially homologous” it is meant that the degree of sequenceidentity of neurturin orthologs including human and mouse neurturin aswell as neurturin from any other species or the degree of sequenceidentity of persephin orthologs including human, mouse and rat persephinas well as persephin from any other species, is greater than thatbetween paralogs such as persephin and neurturin or persephin and GDNF,and greater than that reported previously for members of the TGF-βsuperfamily (For discussion of homology of TGF-β superfamily members seeKingsley, Genes and Dev 8:133-46, 1994 which is incorporated byreference).

Sequence identity or percent identity is intended to mean the percentageof some residues between two sequences. The reference sequence is mousepersephin when determining percent identity with mouse GDNF and mouseneurturin and rat persephin when determining percent identity with ratGDNF and rat neurturin. Referencing is to human neurturin whendetermining percent identity with non-human neurturin, to humanneurturin when determining percent identity with non-neurturin growthfactors and to human GDNF when determining percent identity ofnon-neurturin growth factors with GDNF. In all of the above comparisons,the two sequences being compared are aligned using the Clustal method(Higgins et al, Cabios 8:189-191, 1992) of multiple sequence alignmentin the Lasergene biocomputing software (DNASTAR, INC, Madison, Wis.). Inthis method, multiple alignments are carried out in a progressivemanner, in which larger and larger alignment groups are assembled usingsimilarity scores calculated from a series of pairwide alignments.Optimal sequence alignments are obtained by finding the maximumalignment score, which is the average of all scores between the separateresidues in the alignment, determined from a residue weight tablerepresenting the probability of a given amino acid change occurring intwo related proteins over a given evolutionary interval. Penalties foropening and lengthening gaps in the alignment contribute to the score.The default parameters used with this program are as follows: gappenalty for multiple alignment=10; gap length penalty for multiplealignment=10; k-tuple value in pairwise alignment=1; gap penalty inpairwise alignment=3; window value in pairwise alignment=5; diagonalssaved in pairwise alignment=5. The residue weight table used for thealignment program is PAM250 (Dayhoff et al., in Atlas of ProteinSequence and Structure, Dayhoff, Ed., NBRF, Washington, Vol. 5, suppl.3, p. 345, 1978).

Percent conservation is calculated from the above alignment by addingthe percentage of identical residues to the percentage of positions atwhich the two residues represent a conservative substitution (defined ashaving a log odds value of greater than or equal to 0.3 in the PAM250residue weight table). Conservation is referenced to mouse persephinwhen determining percent conservation with persephin from other speciesor with non-persephin growth factors; referenced to human neurturin whendetermining percent conservation with non-human neurturin or withnon-neurturin growth factors, and eferenced to human GDNF whendetermining percent conservation to non-persephin, non-neurturin growthfactors with GDNF. Conservative amino acid changes satisfying thisrequirement are: R-K; E-D, Y-F, L-M; V-I, Q-H.

Table 2 shows the calculations of identity (I) and conservation (C) forcomparisons of persephin and neurturin and GDNF from various species.Comparisons were made between mouse persephin from the first canonicalframework cysteine to the end (SEQ ID NO:89) and rat persephin from thefirst canonical framework cysteine to the end (SEQ ID NO:83); betweenmouse persephin and mouse GDNF from the first cysteine to the end(mGDNF/C-END, SEQ ID NO:87) or mouse neurturin from the first cysteineto the end (MNTN/C-END, SEQ ID NO:88); and between rat persephin and ratGDNF from the first cysteine to the end (rGDNF/C-End). Neurturincomparisons were between mature human and mature mouse neurturin (hNTNand mNTN, respectively) and between each of these and mature human, ratand mouse GDNF (hGDNF, rGDNF and mGDNF, respectively) as shown in thetable.

TABLE 2 COMPARISON % IDENTITY % CONSERVATION mPSP v. rPSP 96 98 mPSP v.mNTN/C-END 51 54 mPSP v. mGDNF/C-END 41 46 rPSP v. rGDNF/C-END 42 45hNTN v. mNTN 90 93 hNTN v. rGDNF 44 53 hNTN v. mGDNF 43 52 hNTN v. hGDNF43 53 mNTN v. rGDNF 42 52 mNTN v. mGDNF 41 51 mNTN v. hGDNF 41 52

The degree of homology between the mouse persephin and rat persephin isabout 96% and it is believed that the degree of homology between eithermouse or rat persephin and human persephin is at least about 85%identity based upon a similar comparison with neurturin. The neurturincomparisons as shown in Table 2 indicate mature mouse and humanneurturin proteins have about 90% sequence identity. Furthermore, allpersephin and neurturin homologs of non-human mammalian species arebelieved to similarly have at least about 85% sequence identity withhuman persephin and neurturin, respectively. For non-mammalian speciessuch as avian species, it is believed that the degree of homology withpersephin or neurturin is at least about 65% identity with humanpersephin or human neurturin, respectively. By way of comparison, thevariations between family members of the neurturin-persephin-GDNF familyof growth factors can be seen by the comparison of persephin and GDNF orneurturin and GDNF. Mouse and rat persephin have about 35 to 40%sequence identity with mouse and rat GDNF respectively. Similarly, humanand mouse neurturin have about 40% sequence identity and about 50%sequence conservation with human, mouse and rat GDNF. It is believedthat the different family members also have a similar sequence identityof about 40% of that of neurturin, about 40 of that of persephin andabout 40% of that of GDNF and within a range of about 30% to about 85%identity with neurturin, within a range of about 30% to about 85%identity with persephin and within a range of about 30% to about 85%sequence identity with GDNF. Thus, a given member of theGDNF-neurturin-persephin family would be expected to have lessersequence identity with any other family member of the same species thanis present in orthologs of that family member in other species just ashuman GDNF and human neurturin are more closely related to mouse GDNFand mouse neurturin, respectively, than to each other or to GDNF and anygiven family member would be expected to have greater sequence identitywith another family member than to any other known member of the TGF-βsuperfamily (Kingsley, supra).

In the case of pre-pro neurturin, homologs of pre-pro neurturin innon-human mammalian species can be identified by virtue of the neurturinportion of the amino acid sequence having at least about 85% sequenceidentity with human neurturin and homologs of pre-pro neurturin innon-mammalian species can be identified by virtue of the neurturinportion of the amino acid sequence having at least about 65% identitywith human neurturin. It is similarly believed that mammalian pre-propersephin proteins including the human ortholog will have at least about85% sequence identity in the mature persephin portion of the moleculeand that non-mammalian pre-pro persephin proteins will have at leastabout 65% sequence identity with human pre-pro persephin.

Either persephin or neurturin, as the terms are used herein, can alsoinclude hybrid and modified forms of persephin or neurturin,respectively, including fusion proteins and persephin or neurturinfragments and hybrid and modified forms in which certain amino acidshave been deleted or replaced and modifications such as where one ormore amino acids have been changed to a modified amino acid or unusualamino acid and modifications such as glycosolations so long as thehybrid or modified form retains the biological activity of persephin orneurturin. By retaining the biological activity, it is meant thatneuronal survival is promoted, although not necessarily at the samelevel of potency as that of the neurturin isolated from CHO cellconditioned medium or that of the recombinantly produced human or mouseneurturin or human or mouse or rat persephin.

Also included within the meaning of substantially homologous is anypersephin or neurturin which may be isolated by virtue ofcross-reactivity with antibodies to the persephin or neurturin,respectively, as described herein or whose encoding nucleotide sequencesincluding genomic DNA, mRNA or cDNA may be isolated throughhybridization with the complementary sequence of genomic or subgenomicnucleotide sequences of cDNA of the persephin or neurturin,respectively, as described herein or fragments thereof. It will also beappreciated by one skilled in the art that degenerate DNA sequences canencode human neurturin or human persephin and these are also intended tobe included within the present invention as are allelic variants ofneurturin or persephin, respectively.

In the case of pre-pro neurturin, alternatively spliced protein productsresulting from an intron located in the coding sequence of the proregion may exist. The intron is believed to exist in the genomicsequence at a position corresponding to that between nucleic acids 169and 170 of the cDNA which, in turn, corresponds to a position withinamino acid 57 in both the mouse and human pre-pro neurturin sequences(see FIGS. 7 and 8). Thus, alternative splicing at this position mightproduce a sequence that differs from that identified herein for humanand mouse pre-pro neurturin (SEQ ID NO:11 and SEQ ID NO:12,respectively) at the identified amino acid site by addition and/ordeletion of one or more amino acids. Any and all alternatively splicedpre-pro neurturin proteins are intended to be included within the termspre-pro neurturin as used herein.

Although it is not intended that the inventors herein be bound by anytheory, it is thought that the human and mouse proteins identifiedherein as well as homologs from other tissues and species may exist asdimers in their biologically active form in a manner consistent withwhat is known for other factors of the TGF-β superfamily.

In addition to homodimers, the monomeric units of the dimers ofneurturin or persephin can be used to construct stable growth factorheterodimers or heteromultimers comprising at least one monomer unitderived from persephin or at least one monomer unit derived fromneurturin. This can be done by dissociating a homodimer of neurturin ora homodimer of persephin into its component monomeric units andreassociating in the presence of a monomeric unit of a second orsubsequent homodimeric growth factor. This second or subsequenthomodimeric growth factor can be selected from a variety of growthfactors including neurturin, persephin, GDNF, a member of the NGF familysuch as NGF, BDNF, NT-3 and NT-4/5, a member of the TGF-β superfamily, avascular endothelial growth factor, a member of the CNTF/LIF family andthe like.

Growth factors are thought to act at specific receptors. For example,the receptors for TGF-β and activins have been identified and make up afamily of Ser/Thr kinase transmembrane proteins (Kingsley, Genes and Dev8:133-146, 1994; Bexk et al Nature 373:339-341, 1995 which areincorporated by reference). In the NGF family, NGF binds to the TrkAreceptor in peripheral sensory and sympathetic neurons and in basalforebrain neurons; BDNF and NT-4/5 bind to trkB receptors; and NT-3binds primarily to trkC receptors that possess a distinct distributionwithin the CNS (Tuszynski et al., Ann Neurol 35:S9-S12, 1994). Theinventors herein believe that persephin, neurturin, GDNF and as yetunknown members of this family of growth factors act through specificreceptors having distinct distributions as has been shown for othergrowth factor families. These may be separate receptors or it is alsopossible that members of the GDNF-neurturin-persephin family may actupon the same receptor as is the case with BDNF and NT-4/5 which act onthe trkB receptor. Nevertheless, by forming heterodimers orheteromultimers of persephin or neurturin and one or more other growthfactors, the resultant growth factor would be expected to be able tobind to at least two distinct receptor types preferentially having adifferent tissue distribution. The resultant heterodimers orheteromultimers would be expected to show an enlarged spectrum of cellsupon which it could act or provide greater potency. It is also possiblethat the heterodimer or heteromultimer might provide synergistic effectsnot seen with homodimers or homomultimers. For example, the combinationof factors from different classes has been shown to promote long-termsurvival of oligodendrocytes whereas single factors or combinations offactors within the same class promoted short-term survival (Barres etal., Development 118:283-295, 1993).

Heterodimers can be formed by a number of methods. For example,homodimers can be mixed and subjected to conditions in whichdissociation/unfolding occurs, such as in the presence of adissociation/unfolding agent, followed by subjection to conditions whichallow monomer reassociation and formation of heterodimers.Dissociation/unfolding agents include any agent known to promote thedissociation of proteins. Such agents include, but are not limited to,guanidine hydrochloride, urea, potassium thiocyanate, pH lowering agentssuch as buffered HCl solutions, and polar, water miscible organicsolvents such as acetonitrile or alcohols such as propanol oriaopropanol. In addition, for homodimers linked covalently by disulfidebonds as is the case with TGP-β family members, reducing agents such asdithiothreitol and β-mercaptosthanol can be used fordissociation/unfolding and for reassociation/refolding.

Heterodimers can also be made by transfecting a cell with two or morefactors such that the transformed cell produces heterodimers as has beendone with the neurotrophins. (Heymach and Schooter, J Biol Chem270:12297-12304, 1995).

Another method of forming heterodimers is by combining persephin orneurturin homodimers and a homodimer from a second growth factor andincubating the mixture at 37° C.

When heterodimers are produced from homodimers, the heterodimers maythen be separated from homodimers using methods available to thoseskilled in the art such as, for example, by elution from preparative,non-denaturing polyacrylamide gels. Alternatively, heterodimers may bepurified using high pressure cation exchange chromatography such as witha Mono S cation exchange column or by sequential immunoaffinity columns.

It is well known in the art that many proteins are synthesized within acell with a signal sequence at the N-terminus of the mature proteinsequence and the protein carrying such a leader sequence is referred toas a preprotein. The pre- portion of the protein is cleaved duringcellular processing of the protein. In addition to a pre- leadersequence, many proteins contain a distinct pro sequence that describes aregion on a protein that is a stable precursor of the mature protein.Proteins synthesized with both pre- and pro- regions are referred to aspreproproteins. In view of the processing events known to occur withother TGF-β family members as well as the sequences determined herein,the inventors believe that the forms of persephin or neurturin proteinas synthesized within a call in the pre-pro persephin or a pre-proneurturin. In the case of neurturin, the pre-pro neurturin is believedto contain an N-terminal 19 amino acid signal sequence (human pre-signal sequence, SEQ ID NO:15, FIG. 7, amino acids 1 through 19 encodedby SEQ ID NO:17, FIG. 7 , nucleic acids 1 through 57; mouse pre- signalsequence, SEQ ID NO:16, FIG. 8, amino acids 1 through 19, encoded by SEQID NO:18, FIG. 8, nucleic acids 1 through 57). It is known that the fulllength of a leader sequence is not necessarily required for the sequenceto act as a signal sequence and, therefore, within the definition ofpre- region of neurturin is included fragments thereof, usuallyN-terminal fragments, that retain the property of being able to act as asignal sequence, that is to facilitate co-translational insertion intothe membranes of one or more cellular organelles such as endoplasmicreticulum, mitochondria, golgi, plasma membrane and the like.

The neurturin signal sequence is followed by a pro-domain which containsan RXXR proteolytic processing site immediately before the N-terminalamino acid sequence for the mature neurturin. (human pro- regionsequence, SEQ ID NO:19, FIG. 7, amino acids 20 through 95 encoded by thenucleic acid sequence SEQ ID NO:20, FIG. 7 nucleic acids 58 through 285;mouse pro- region sequence, SEQ ID NO:22, FIG. 8, amino acids 19 through95 encoded by nucleic acid sequence SEQ ID NO:21, FIG. 8 , nucleic acids58 through 285).

The neurturin pre- and pro- regions together comprise a pre-pro sequenceidentified as the human pre-pro sequence (SEQ ID NO:23, FIG. 7 , aminoacids 1 through 95 encoded by SEQ ID NO:25, nucleic acids 1 through 285)and the mouse pre-pro sequence (SEQ ID NO:24, FIG. 8, amino acids 1through 95 encoded by SEQ ID NO:26, nucleic acids 1 through 285). Thepre- region sequences and pro- region sequences as well as the pre-proregion sequences can be identified and obtained for non-human mammalianspecies and for non-mammalian species by virtue of the sequences beingcontained within the pre-pro neurturin as defined herein. It is believedthat persephin is similarly associated with pre- end pro- regions toconstitute a pre-pro persephin sequence.

Using the above landmarks, the mature, secreted neurturin molecule ispredicted to be approximately 11.5 kD which is likely to form adisulfide linked homodimer of approximately 23 kD by analogy to othermembers of the TGF-β family. The predicted approximately 23 kD proteinis consistent with the 25 kD neurturin protein purified from CHO cellconditioned media being a homodimer. The inventors herein have detectedan approximately 11.5 kD neurturin protein from conditioned medium ofChinese hamster ovary cells transfected with the neurturin expressionvector (pCMV-NTN-3-1) using SDS-PAGE under reducing conditions and thisprotein is thought to be the monomer.

As discussed above, a mature persephin molecule predicted on the basisof homology to neurturin would contain 5 amino acids upstream from thefirst canonical framework cysteine thus having 96 amino acids and apredicted molecular mass of 10.4 kD. A mature persephin molecule basedupon homology to GDNF would contain 43 amino acids upstream from thefirst canonical framework cysteine thus having 134 amino acids and apredicted molecular mass of 14.5 kD.

The nucleotide sequences of neurturin pre- and/or pro- regions orsimilar regions that are believed to be associated with persephin DNAcan be used to construct chimeric genes with the coding sequences ofother growth factors or proteins and, similarly, chimeric genes can beconstructed from the coding sequence of neurturin coupled to sequencesencoding pre- and/or pro- regions from genes for other growth factors orproteins. (Booth et al., Gene 146:303-8, 1994; Ibanez, Gene 146:303-8,1994; Storici et al., FEBS Letters 337:303-7, 1994; Sha et al J CellBiol 114:827-839, 1991 which are incorporated by reference). Suchchimeric proteins can exhibit altered production or expression of theactive protein species.

A preferred neurturin has been identified and isolated in purified formfrom medium conditioned by CHO cells. Also preferred is neurturinprepared by recombinant DNA technology. Similarly, a preferred persephinaccording to the present invention is prepared by recombinant DNAtechnology.

By “pure form” or “purified form” or “substantially purified form” it ismeant that a persephin or neurturin composition is substantially free ofother proteins which are not persephin or neurturin, respectively.

Recombinant persephin or neurturin may be made by expressing the DNAsequences encoding persephin or neurturin, respectively, in a suitabletransformed host cell. Using methods well known in the art, the DNAencoding persephin or neurturin may be linked to an expression vector,transformed into a host call and conditions established that aresuitable for expression of persephin or neurturin, respectively, by thetransformed cell.

Any suitable expression vector may be employed to produce recombinanthuman persephin or recombinant human neurturin such as, for example, themammalian expression vector pCB6 (Brewer, Meth Cell Biol 43:233-245,1994) or the E. coli pET expression vectors, specifically, pET-30a(Studier et al., Methods Enzymol 185:60-89, 1990 which is incorporatedby reference) both of which were used herein. Other suitable expressionvectors for expression in mammalian and bacterial cells are known in theart as are expression vectors for use in yeast or insect cells.Baculovirus expression systems can also be employed.

Persephin or neurturin may be expressed in the monomeric units or suchmonomeric form may be produced by preparation under reducing conditions.In such instances refolding and renaturation can be accomplished usingone of the agents noted above that is known to promotedissociation/association of proteins. For example, the monomeric formcan be incubated with dithiothreitol followed by incubation withoxidized glutathione disodium salt followed by incubation with a buffercontaining a refolding agent such as urea.

In the case of neurturin, by analogy with the N-terminal sequence andinternal fragments of the neurturin purified from CHO call conditionedmedium, the mature mouse sequence was deduced and from this the maturehuman form was predicted using the sequence from the human gene. Theamino acid sequence of the mature human form is as shown in FIG. 5(hNTN, SEQ ID NO:1). The material purified from CHO call conditionedmedium is considered to be mature neurturin and may exist as a dimer orother multimer and may be glycosylated or chemically modified in otherways.

Persephin, like neurturin, may also exist as a dimer or other multimerand may be glycosylated or chemically modified in other ways.

As noted above, the mouse and human nucleic acid sequences suggest thatneurturin is initially translated as a pre-pro polypeptide and thatproteolytic processing of the signal sequence and the “pro” portion ofthis molecule results in the mature sequence, referenced herein as“mature neurturin”, as obtained from medium condition by CHO cells andas exists in human and in non-human species in homologous form.Neurturin, therefore, includes any and all “mature neurturin” sequencesfrom human and non-human species and any and all pre-pro neurturinpolypeptides that may be translated from the neurturin gene.

As with neurturin, the persephin in the present invention also includesany and all mature persephin sequences from human and non-human speciesand any and all pre-pro persephin polypeptides that may be translatedfrom the persephin gene.

It is believed that the coding sequence for the pre-pro-neurturinpolypeptide begins at the first ATG codon encoding methionine at the 5′end of the clone (position 1 in FIG. 9) which is positioned in the samereading frame as the sequence encoding the amino acid sequences obtainedfrom the purified neurturin. Downstream from the first codon is thelargest open reading frame containing the coding sequence for the pre-and pro-regions followed by the coding sequence for the mature mouseneurturin.

Sequence analysis of the murine neurturin genomic clones identified a0.5 kb intron located between nucleotide 169 and 170 of the pre-proneurturin from the cDNA clones. This intron is located in the codingsequence of the pro- region of the pre-pro-neurturin protein. Thus, itis believed that the mouse neurturin gene contains at least two exons,one of which contains the coding sequences upstream from the splice siteand the other contains the coding sequence downstream (FIG. 8, SEQ IDNO:29, SEQ ID NO: 30). It is known that the gene for GDNF contains anintron located at an analogous position and an alternately spliced formof GDNF has been detected by RT-PCR experiments (Suter-Crazzolare andUnsicker, Neuroreport 5: 2486-2488, 1994 which is incorporated byreference). This alternate form results from the use of a splice site inthe second coding axon located 78 by 3′ to the original splice sitereported. The alternately spliced form encodes a GDNF protein with adeletion of 26 amino acids relative to the originally reported form. Thetwo forms are expressed in different ratios in different tissues. Wehave not detected alternately spliced forms of neurturin in RT-PCR andRACE experiments using mouse P1 brain and P2 liver cDNAs. Thepossibility exists, however, that alternate splice sites in theneurturin gene may be utilized in different tissues.

The coding sequence of the human neurturin cDNA has been deduced fromthe sequence of the human neurturin genomic clones. The coding sequenceof the human cDNA, like that of the mouse cDNA, is interrupted by anintron between nucleotides 169 and 170 of the coding sequence. Thus, thehuman neurturin gene is believed to contain at least two exons, one ofwhich contains the coding sequence upstream from the splice site end theother contains the coding sequence downstream (FIG. 7, SEQ ID NO:27, SEQID NO:28). The splice sites at the intron-exon junctions of the humanand mouse genes have been conserved.

From the deduced amino acid sequence of human neurturin, the earlierpredicted N-terminal sequence lies between positions 286 end 339 and thepredicted internal sequences lie between positions 385 and 417,positions 474 and 533 and positions 547 and 576. The TGA stop codon atpositions 592-594 terminate the open reading frame.

The predicted length of the purified pre-pro neurturin is 197 amino acidresidues for the human pre-pro neurturin (SEQ ID NO:7) and 195 aminoacid residues for the mouse pre-pro neurturin (SEQ ID NO:8). Thepredicted molecular weight of this polypeptide is 22.2 kD for mouse and22.4 kd for human. The predicted length of the purified neurturin is 100amino acid residues end its predicted monomeric molecular weight is 11.5kD. There are no N-linked glycosylation sites, however, potential0-linked glycosolation sites occur at amino acid residues in positions18, 26, 80, 86 and 95 in human neurturin. Glycosylation at any one orcombination of these sites would increase the molecular weight of themolecule.

In the case of persephin, there are no N-linked glycosylation sites inthe region between the first and seventh canonical framework cysteines(SEQ ID NO:79) nor are there any N-linked glyosylation sites in a maturepersephin molecule predicted on the basis of homology to neurturin (SEQID NO:80). In a mature persephin molecule based upon homology to GDNFthere are two potential N-linked glycosylation sites in the 43 aminoacids upstream from the first canonical framework cysteine at positions31 and 32 in SEQ ID NO:81 (corresponding to positions 39 and 40 in thesequence as shown in FIG. 11).

Potential 0-linked glycosylation sites occur in persephin in the regionbetween the first and seventh canonical framework cysteines at positions5, 7, 19, 31, 38, 41, 62, 63, 68 and 83 in SEQ ID NO:79 (FIG. 12) and ina mature persephin molecule predicted on the basis of homology toneurturin (SEQ ID NO:80) there is one additional potential 0-linkedglycosylation site one residue upstream from the first canonicalframework cysteine (position 51 in the sequence an shown in FIG. 11). Ina mature persephin molecule based upon homology to GDNF there are fivepotential 0-linked glycosylation sites in the 43 amino acids upstreamfrom the first canonical framework cysteine at positions 9, 15, 18, 22and 43 in SEQ ID NO:81 (corresponding to positions 17, 23, 26, 30 and 51in the sequence as shown in FIG. 11) along with the ten potential0-linked glycosylation sites noted above in the region between thecanonical framework cysteines (corresponding to positions 48, 50, 62,74, 81, 84, 105, 106, 111 and 126 in SEQ ID NO:81 and positions 56, 58,70, 82, 89, 92, 113, 114, 119 and 134 in the sequence as shown in FIG.11).

Different possible cleavage sites may be present in thepre-pro-neurturin sequence. The amino acid sequence of the mature mouseneurturin (FIG. 5 , SEQ ID NO:2) is predicted from alignment with theN-terminal amino acid sequence of the purified Chinese hamsterneurturin. A four residue RRAR cleavage site (amino acids 92-95) isfound immediately before the predicted N-terminal amino acid of maturemouse neurturin. This RRAR sequence fits the RXXR consensus sequence atwhich members of the TGF-β superfamily are usually cleaved. Thisputative RRAR cleavage sequence is conserved in human neurturin.However, the mature human neurturin is predicted to have a two aminoacid N-terminal extension relative to mature mouse neurturin whencleaved at this sequence. Since neurturin contains other sequences whichfit the RXXR consensus (for example the sequence RRRR at amino acids90-93) and the specificities of proteases involved in this cleavage arenot completely understood, the possibility exists that in somesituations, neurturin is cleaved at sites other than the above RRARsequence, and the mature neurturin protein may have a variable number ofamino acids preceding the cysteine residue at position 101 in the mousesequence (pre-pro protein) and position 103 in the human sequence. Suchalternate cleavage sites could be utilized differently among differentorganisms and among different tissues of the same organism. TheN-terminal amino acids preceding the first of the seven conservedcysteines in the mature forms of members of the TGF-β family varygreatly in both length and sequence. Furthermore, insertion of a tenamino acid sequence two residues upstream of the first conservedcysteine does not affect the known biological activities of one familymember, dorealin (Basler, K., Edlund, T., Jessell, T. M., and Yamada,T., (1993) Cell 73:687-702). Thus neurturin proteins which containsequences of different lengths preceding the cysteine 101 in mouse andcysteine 103 in human would be likely to retain their biologicalactivity.

It is also believed that persephin proteins which contain sequences ofdifferent lengths preceding the first cysteine (residue No. 1 of mousepersephin in FIG. 12 and residue No. 1 of rat persephin in FIG. 14)would be likely to retain their biological activity.

The inventors herein believe that at a minimum the sequence of neurturinthat will show biological activity will contain the sequence beginningat cysteine 103 and ending at cysteine 196 for human neurturin (FIG. 7 ,SEQ ID NO:31) and beginning at cysteine 101 and ending at cysteine 194for mouse neurturin (FIG. 7 , SEQ ID NO:32). Thus, within the scope ofthe neurturin polypeptides are amino acid sequences containing SEQ IDNO:31 and amino acid sequences containing SEQ ID NO:32 and nucleic acidsequences encoding these amino acid sequences.

Similarly, the inventors herein believe that, at a minimum, the sequenceof persephin that will show biological activity will contain thesequence beginning at cysteine 1 and ending at cysteine 87 for mousepersephin (FIG. 12 , SEQ ID NO:79) and beginning at cysteine 1 andending et cysteine 87 for rat persephin (FIG. 14 , SEQ ID NO:82). Thus,within the scope of persephin of the present invention are amino acidsequences containing SEQ ID NO:79 and amino acid sequences containingSEQ ID NO:82 and nucleic acid sequences encoding these amino acidsequences.

The present invention also encompasses nucleic acid sequences includingsequences that encode mouse and rat persephin (FIGS. 11 and 14) as wellas human persephin in the same manner that neurturin includes human endmouse neurturin nucleic acid sequences (FIGS. 7 and 8). Also includedwithin the scope of this invention are sequences that are substantiallythe same as the nucleic acid sequences encoding persephin or neurturin,respectively. Such substantially the same sequences may, for example, besubstituted with codons more readily expressed in a given host call suchas E. coli according to well known and standard procedures. Suchmodified nucleic acid sequences are included within the scope of thisinvention.

Specific nucleic acid sequences can be modified by those skilled in theart and, thus, all nucleic acid sequences which encode for the aminoacid sequences of pre-pro neurturin or persephin or the pre- region orthe pro- region of neurturin or persephin can likewise be so modified.The present invention thus also includes nucleic acid sequence whichwill hybridize with all such nucleic acid sequences—or complements ofthe nucleic acid sequences where appropriate—and encode for apolypeptide having cell survival or growth promoting activity. Thepresent invention also includes nucleic acid sequences which encode forpolypeptides that have survival or growth promoting activity and thatare recognized by antibodies that bind to neurturin or by antibodiesthat bind to persephin.

The present invention also encompasses vectors comprising expressionregulatory elements operably linked to any of the nucleic acid sequencesincluded within the scope of the invention. This invention also includeshost cells—of any variety—that have been transformed with vectorscomprising expression regulatory elements operably linked to any of thenucleic acid sequences included within the scope of the presentinvention.

Methods are also provided herein for producing neurturin or persephin.Preparation can be by isolation from conditioned medium from a varietyof cell types so long am the cell type produces neurturin or persephin.A second and preferred method involves utilization of recombinantmethods by isolating a nucleic acid sequence encoding neurturin orpersephin, cloning the sequence along with appropriate regulatorysequences into suitable vectors and cell types, and expressing thesequence to produce neurturin or persephin.

A mammalian gene family comprised of four neurotrophic factors has beenidentified including nerve growth factor (NGF), brain derivedneurotrophic factor (BDGF), neurotrophin-3 (NT-3), and neurotrophin-4/5(NT-4/5). These factors share approximately 60 percent nucleic acidsequence homology (Tuezynaki and Gage, Ann Neurol 35:S9--12, 1994 whichis incorporated by reference). The persephin protein and the neurturinprotein display no significant homology to the NGF family ofneurotrophic factors. Either persephin or neurturin shares lose thanabout 208 homology with the TGF-β superfamily of growth factors.However, both persephin and neurturin show approximately 40% sequenceidentity with GDNF and approximately 50% sequence identity with eachother. In particular, the positions of the seven cysteine residuespresent in persephin, neurturin and GDNF are nearly exactly conserved.The inventors herein believe that other unidentified genes may existthat encode proteins that have substantial amino acid sequence homologyto persephin, neurturin and GDNF and which function an growth factorsselective for the same or different tissues end the same or differentbiological activities and may act at the same or different receptors. Adifferent spectrum of activity with respect to tissues affected and/orresponse elicited could result from preferential activation of differentreceptors by different family members as is known to occur with membersof the NGF family of neurotrophic factors (Tuszynski and Gage, 1994,supra).

As a consequence of members of a particular gene family showingsubstantial conservation of amino acid sequence among the proteinproducts of the family members, there is considerable conservation ofsequences at the DNA level. This forms the basis for a new approach foridentifying other members of the gene family to which GDNF, neurturinand persephin belong. The method used for such identification iscross-hybridization using nucleic acid probes derived from one familymember to form a stable hybrid duplex molecule with nucleic acidsequence from different members of the gene family or to amplify nucleicacid sequences from different family members. (see for example, Kaishoet al. FEBS Letters 266:187-191, 1990 which is incorporated byreference). The sequence from the different family member may not beidentical to the probe, but will, nevertheless be sufficiently relatedto the probe sequence to hybridize with the probe. Alternatively, PCRusing primers from one family member can be used to identify additionalfamily members.

The above approaches have not heretofore been successful in identifyingother gene family members because only one family member, GDNF wasknown. With the identification of neurturin in copending applicationSer. No. 08/519,777, however, unique new probes and primers can be madethat contain sequences from the conserved regions of this gene family.The same conserved regions are also found in the third family member,persephin. In particular, three conserved regions have been identifiedherein which can be used as a basis for constructing new probes andprimers. The new probes and primers made available from the work withneurturin and persephin make possible this powerful new approach whichcan now successfully identify other gene family members. Using this newapproach, one may screen for genes related to GDNF, neurturin andpersephin in sequence homology by preparing DNA or RNA probes based uponthe conserved regions in the GDNF and neurturin molecules. Therefore,one embodiment of the present invention comprises probes and primersthat are unique to or derived from a nucleotide sequence encoding suchconserved regions and a method for identifying further members of theneurturin-persephin-GDNF gene family.

Conserved-region amino acid sequences have been identified herein toinclude Val-Xaa₁- Xaa₂-Leu-Gly-Leu-Gly-Tyr where Xaa₁ is Ser, Thr or Alaand Xaa₂ is Glu or Asp (SEQ ID NO:108);Glu-Xaa₁-Xaa₂-Xaa₃-Phe-Arg-Tyr-Cys-Xaa₄-Gly-Xaa₅-Cys in which Xaa₁ isThr, Glu or lys, Xaa₂ is Val, Leu or Ile, Xaa₃ is Leu or Ile, Xaa₄ isAla or Ser, and Xaa₅ is Ala or Ser, (SEQ ID NO:113); andCys-Cys-Xaa₁-Pro-Xaa₂-Xaa₃-Xaa₄-Xaa₅-Asp-Xaa₆-Xaa₇-Xaa₈-Phe-Leu-Asp-Xaa₉in which Xaa₁ is Arg or Gln, Xaa₂ is Thr or Val or Ile, Xaa₃ is Ala orSer, Xaa₄ is Tyr or Phe, Xaa₅ is Glu, Asp or Ala, Xaa₆ is Glu, Asp or noamino acid, Xaa, in val or leu, Xaa₈ is Ser or Thr, and Xaa₉ is Asp orVal (SEQ ID NO:114). Nucleotide sequences containing a coding sequencefor the above conserved sequences or fragments of the above conservedsequences can be used as probes. Exemplary probe and primer sequencesencoding amino acid sequences end SEQ ID NOS:125-129; primers whosereverse complementary sequences encode amino acid sequences SEQ IDNO:126, SEQ ID NO:127, SEQ ID NO:130; and, in particular, nucleotidesequences, SEQ ID NOS:115-124. Additional primers based upon GDNF andneurturin include nucleic acid sequences encoding amino acid sequences,SEQ ID NO:33, SEQ ID NO:36, SEQ ID NO:40 and SEQ ID NO:41; primers whosereverse complementary sequences encode SEQ ID NO:37, SEQ ID NO:38 andSEQ ID NO:39; and, in particular, nucleic acid sequences, SEQ IDNOS:42-48.

Hybridization using the new probes from conserved regions of the nucleicacid sequences would be performed under reduced stringency conditions.Factors involved in determining stringency conditions are wall known inthe art (for example, see Sambrook et al., Molecular Cloning, 2nd Ed.,1989 which is incorporated by reference). Sources of nucleic acid forscreening would include genomic DNA libraries from mammalian species orcDNA libraries constructed using RNA obtained from mammalian cellscloned into any suitable vector.

PCR primers would be utilized under PCR conditions of reduced annealingtemperature which would allow amplification of sequences from genefamily members other than GDNF, neurturin and persephin. Sources ofnucleic acid for screening would include genomic DNA libraries frommammalian species cloned into any suitable vector, cDNA transcribed fromRNA obtained from mammalian cells, and genomic DNA from mammalianspecies.

DNA sequences identified on the basis of hybridization or PCR assayswould be sequenced and compared to GDNF, neurturin and persephin. TheDNA sequences encoding the entire sequence of the novel factor wouldthen be obtained in the same manner as described herein. Genomic DNA orlibraries of genomic clones can also be used as templates because theintron/axon structures of GDNF and neurturin are conserved and codingsequences of the mature proteins are not interrupted by introns.

Using this approach as described above, the primers designed from theconserved regions of neurturin and GDNF have been used to identify andobtain the sequence of the new family member described herein,persephin. Degenerate primers designed from persephin, neurturin andGDNF can be further used to identify and obtain additional familymembers.

It is believed that all GDNF-neurturin-persephin family members willhave a high degree of sequence identity with one or more of the threeidentified family-member consensus regions in the portion of thesequence between the first and seventh canonical framework cysteines(see FIG. 12). In particular, a now family member is anticipated to haveat least a 62.5% identity with the consensus region octapeptide,Val-Xaa₁-Xaa₂-Leu-Gly-Leu-Gly-Tyr where Xaa₁ is Ser, Thr or Ala and Xaa₂is Glu or Asp (SEQ ID NO:108) or at least a 62.5 percent sequenceidentity with the consensus region octapeptide,Phe-Arg-Tyr-Cys-Xaa₁-Gly-Xaa₂-Cys where Xaa₁ and Xaa₂ are alanine orserine (SEQ ID NO:109) or at least a 50 percent sequence identity withthe consensus region octapeptide, Asp-Xaa₁-Xaa₂-Xaa₃-Phe-Leu-Asp-Xaa₄where Xaa₁ is aspartic acid or glutamic acid or no amino acid, Xaa₂ isvaline or leucine, Xaa₃ is serine or threonine; and Xaa₄ is valine oraspartic acid (SEQ ID NO:110). The inventors herein believed that anynew family member will have 28 amino acids in the aligned sequencebetween the first and seventh canonical framework cysteine residues asset forth in FIG. 15 with residues numbered from the N-terminal end ofthe family member aligned sequence being (1) Cys, (3) Leu, (10) Val,(13) Leu, (14) Gly, (15) Leu, (16) Gly, (17) Tyr, (21) Glu, (25) Phe,(26) Arg, (27) Tyr, (28) Cys, (30) Gly, (32) Cys, (44) Leu, (47) Leu,(58) Cys, (59) Cys (61) Pro, (66) Asp. (69) Phe, (70) Leu, (71) Asp,(83) Ser, (84) Ala, (87) Cys, and (89) Cys, however, it is possible thatthere may be as many as three mismatches.

Although neurturin has been purified on the basis of its ability topromote the survival of a particular neuronal type, this factor will acton other neuronal cell types as well. For example, neurturin is shownherein to promote the survival of nodose sensory ganglia neurons (seaExample 3). Neurturin is also likely to promote the survival ofnon-neuronal cells. Indeed, all the growth factors isolated to data havebeen shown to act on many different cell types (for example see Scullyand Otten, Cell Biol Int 19:459-469, 1005; Hefti, Neurotrophic FactorTherapy 25:1418-1435, 1994 which are incorporated by reference). It isknown that NGF acts on sympathetic neurons, several types of sensoryneurons and certain populations of CNS neurons. GDNF, which is moreclosely related to neurturin, has been shown to act on dopaminergic,sympathetic, motor and several sensory neurons (Henderson et al. Supra,1994: Miles et al, J Cell Biol 130:137-148, 1995; Yan et al, Nature373:341-344, 1995; Lin et al, Science 260:1130-1132, 1993; Trupp et al,J Cell 8101 130:137-148, 1995; Martin et al Brain Res 683:172-178, 1995;Bowenkamp et al J Comp Neurol 355:479-489, 1995 which are incorporatedby reference). Thus, it is likely that in addition to peripheralsympathetic and sensory neurons, neurturin can act on a wide variety ofcentral and peripheral neuronal call types.

On the basis of the structural similarities of persephin to thesequences of neurturin and GDNF, persephin is also believed to promotethe survival and growth of neuronal as wall as non-neuronal cells.Indeed, as noted above, all the growth factors isolated to data havebeen shown to act on many different cell types (Scully and Otten, CellBiol Int 19:459-469, 1005; Hefti, Neurotrophic Factor Therapy25:1418-1435, 1994). Furthermore, the inventors herein have identifiedbrain and heart tissues as tissues expressing persephin, which furthersupports the conclusion that persephin can act to promote survival andgrowth in a variety of neuronal and non-neuronal cells.

As an example of the actions of neurotrophic factors on non-neuronaltissues, the prototypical neurotrophic factor, NGF, also acts upon mastcells to increase their number when injected into newborn rate (Aloe, JNeuroimmunol 18:1-12, 1988). In addition, mast cells express the trkreceptor and respond to NGF such that NGF is a mast cell secretogogueand survival promoting factor (Horigome et al., J Biol Chem269:2695-2707, 1994 which is incorporated by reference). Moreover,members of the TGF-β superfamily act on many cell types of differentfunction and embryologic origin.,

The inventors herein have identified several non-neuronal tissues inwhich neurturin is expressed including blood, bone marrow, neonatalliver and mast cells. This suggests a role for neurturin inhematopoiesis, inflammation, allergy, and cardiomyopathy.

Similarly, the inventors herein have identified brain and heart astissues in which persephin is expressed and it is further believed thatpersephin is expressed in a number of other neuronal and non-neuronaltissues. Thus, persephin may also have a role in hematopoiesis,inflammation, allergy and cardiomyopathies.

Neurotrophic factors of the NGF family are thought to act throughfactor-specific high affinity receptors (Tuszyneki and Gage, 1994,supra). Only particular portions of the protein acting at a receptorSite are required for binding to the receptor. Such particular portionsor discrete fragments can serve as an agonist where the substanceactivates the receptor to elicit the promoting action on call survivaland growth and antagonists to neurturin or persephin where they bind to,but do not activate, the receptor or promote survival and growth. Suchportions or fragments that are agonists and those that are antagonistsare also within the scope of the present invention.

Synthetic, pan-growth factors can also be constructed by combining theactive domains of persephin or neurturin with the active domains of oneor more other growth factors. (For example, see Ilag et al., Proc Nat'lAcad Sci 92:607-611, 1995 which is incorporated by reference). Thesepan-growth factors would be expected to have the combined activities ofneurturin or persephin and the one or more other growth factors. As suchthey are believed to be potent end multispecific growth factors that areuseful in the treatment of a wide spectrum of degenerative diseases andconditions including conditions that can be treated by any and all ofthe parent factors from which the active domains were obtained. Suchpan-growth factors might also provide synergistic effects beyond theactivities of the parent factors (Barrel et al., supra).

Pan-growth factors within the scope of the present invention can alsoinclude chimeric or hybrid polypeptides that are constructed fromportions of fragments of at least two growth factors. Growth factors ofthe TGF-β superfamily are structurally related having highly conservedsequence landmarks whereby family members are identified. In particular,seven canonical framework cysteine residues are nearly invariant inmembers of the superfamily (Kingsley, Genes & Dev 8:133-146, 1994 whichis incorporated by reference) (see FIG. 17). Chimeric polypeptidemolecules can, therefore, be constructed from a sequence that issubstantially identical to a portion of either the persephin or theneurturin molecule, up to one or more crossover points, and one or moresequences each of which is substantially identical with a portion ofanother TGF-β superfamily member extending on the other side of thecorresponding one or more crossover points. For example, a portion ofthe amino terminal and of the persephin polypeptide can be combined witha portion of the carboxy terminal end of a neurturin polypeptide oralternatively a portion of the amino terminal end of a neurturinpolypeptide can be combined with a portion of the carboxy terminal andof a persephin polypeptide. Such portions of neurturin or persephinpolypeptides are preferably from about 5 to about 95, more preferablyfrom about 10 to about 90, still more preferably from about 20 to about80 and most preferably from about 30 to about 70 contiguous amino acidsand such portions of another, non-persephin or, as the case may be,non-neurturin TGF-β superfamily member are preferably from about 5 toabout 95, more preferably from about 10 to about 90, still morepreferably from about 20 to about 80 and most preferably from about 30to about 70 contiguous amino acids. For example, a particular crossoverpoint might be between the third and fourth canonical framework cysteineresidues. One such exemplary construct would contain at the 5′ and asequence comprised of a persephin sequence from residue 1 through thethird canonical framework cysteine residue 37 and up to a cross-overpoint somewhere between residue 37 and residue 63 but not including thefourth canonical framework cysteine residue 64 (for reference, seemature persephin, SEQ ID NO:80). The 3′ end of the hybrid constructwould constitute a sequence derived from another TGF-β superfamilymember such as, for example, neurturin which is another TGF-βsuperfamily member that is closely related to persephin. Using neurturinas the other TGF-β family member, the hybrid construct beyond thecrossover point would be comprised of a sequence beginning at thedesired crossover point in the neurturin sequence between the thirdcanonical framework cysteine residua 37 and the fourth canonicalframework cysteine residue 67 of neurturin and continuing throughresidue 100 at the 3′ end of neurturin (for alignment, see FIG. 12). Asecond exemplary hybrid construct would be comprised of residue 1through a crossover point between residues 37 and 67 of neurturincontiguously linked with residues from the crossover point betweenresidues 37 and 64 through residue 96 of persephin. The above constructswith persephin and neurturin are intended as examples only with theparticular TGF-β family member being selected from family membersincluding but not limited to transforming growth factor-β1 (TGFβ1),transforming growth factor-β2 (TGFβ2), transforming growth factor-β3(TGFβ3), inhibin β A (INHβA), inhibin β B (INHβB), the nodal gene(NODAL), bone morphogenetic proteins 2 and 4 (BMP2 and BMP4), theDrosophila decapentaplegic gene (dpp), bone morphogenetic proteins 5-8(BNP5, BMP6, BMP7 and BMP8), the Drosophila 60A gene family (60A), bonemorphogenetic protein 3 (BMP2), the Vgl gene, growth differentiationfactors 1 and 3 (GDF1 and GDF2), dorsalin (drsln), inhibin α (INHα), theMIS gene (MIS), growth factor 9 (GDF-9), glial-derived neurotropicgrowth factor (GDNF), neurturin (NTN) and persephin (see FIG. 16). Inaddition, the crossover point can be any residue between the first andseventh canonical framework cysteines molecules of neurturin and theparticular other family member. Furthermore, additional crossover pointscan be used to incorporate any desired number of persephin portions orfragments with portions or fragments of any one or more other familymembers.

In constructing a particular chimeric molecule, the portions ofpersephin and portions of the other, non-persephin growth factor areamplified using PCR, mixed and used as template for a PCR reaction usingthe forward primer from one and the reverse primer from the other of thetwo component portions of the chimeric molecule. Thus, for example aforward and reverse primers are selected to amplify the portion ofpersephin from the beginning to the selected crossover point between thethird and fourth canonical cysteine residues using a persephin plasmidas template. A forward primer with a 5′ portion overlapping with thepersephin sequence and a reverse primer are then used to amplify theportion of the other, non-persephin growth factor member of the TGF-βsuperfamily from the corresponding crossover point through the 3′ endusing a plasmid template containing the coding sequence for thenon-persephin TGF-β family member. The products of the two PCR reactionsare gel purified and mixed together and a PCR reaction performed. Usingan aliquot of this reaction as template a PCR reaction is performedusing the persephin forward primer and the reverse primer for thenon-persephin growth factor. The product is then cloned into anexpression vector for production of the chimeric molecule.

Chimeric growth factors would be expected to be effective in promotingthe growth and development of cells and for use in preventing theatrophy, degeneration or death of cells, particular in neurons. Thechimeric polypeptides may also act as a receptor antagonists of one orboth of the full length growth factors from which the chimericpolypeptide was constructed or as an antagonist of any other growthfactor that acts at the same receptor or receptors.

The present invention also includes therapeutic or pharmaceuticalcompositions comprising persephin or neurturin in an effective amountfor treating patients with cellular degeneration or dysfunction and amethod comprising administering a therapeutically effective amount ofneurturin or persephin. These compositions and methods are useful fortreating a number of degenerative diseases. Where the cellulardegeneration involves neuronal degeneration, the diseases include, butare not limited to peripheral neuropathy, amyotrophic lateral sclerosis,Alzheimer's disease, Parkinson's disease, Huntington's disease, ischemicstroke, acute brain injury, acute spinal chord injury, nervous systemtumors, multiple sclerosis, peripheral nerve trauma or injury, exposureto neurotoxins, metabolic diseases such as diabetes or renaldysfunctions and damage caused by infectious agents. In particular, theability of persephin to promote survival in mesencephalic cells suggestsan applicability of this growth factor in treating neuronal degenerativediseases of the CNS such as Parkinson's disease.

Where the cellular degeneration involves bone marrow cell degeneration,the diseases include, but are not limited to disorders of insufficientblood cells such as, for example, leukopenias including eosinopeniaand/or basopenia, lymphopenia, monocytopenia, neutropenia, anemias,thrombocytopenia as well as an insufficiency of stem cells for any ofthe above. The cellular degeneration can also involve myocardial musclecells in diseases such as cardiomyopathy and congestive heart failure.The above cells and tissues can also be treated for depressed function.

The compositions and methods herein can also be useful to preventdegeneration and/or promote survival in other non-neuronal tissues aswell. One skilled in the art can readily determine using a variety ofassays known in the art for identifying whether neurturin or persephinwould be useful in promoting survival or functioning in a particularcell type.

In certain circumstances, it may be desirable to modulate or decreasethe amount of persephin or neurturin expressed. Thus, in another aspectof the present invention, persephin or neurturin anti-senseoligonucleotides can be made and a method utilized for diminishing thelevel of expression of persephin or neurturin, respectively, by a callcomprising administering one or more persephin or neurturin anti-senseoligonucleotides. By persephin or neurturin anti-sense oligonucleotidesreference is made to oligonucleotides that have a nucleotide sequencethat interacts through base pairing with a specific complementarynucleic acid sequence involved in the expression of persephin orneurturin, respectively, such that the expression of persephin orneurturin is reduced. Preferably, the specific nucleic acid sequenceinvolved in the expression of persephin or neurturin is a genomic DNAmolecule or mRNA molecule that contains sequences of the persephin orneurturin gene. This genomic DNA molecule can comprise flanking regionsof the persephin or neurturin gene, untranslated regions of persephin orneurturin mRNA, the pre- or pro- portions of the persephin or neurturingene or the coding sequence for mature persephin or neurturin protein.The term complementary to a nucleotide sequence in the context ofpersephin or neurturin antisense oligonucleotides and methods thereforemeans sufficiently complementary to such a sequence as to allowhybridization to that sequence in a cell, i.e., under physiologicalconditions. The persephin or neurturin antisense oligonucleotidespreferably comprise a sequence containing from about 8 to about 100nucleotides and more preferably the persephin or neurturin antisenseoligonucleotides comprise from about 15 to about 30 nucleotides. Thepersephin or neurturin antisense oligonucleotides can also contain avariety of modifications that confer resistance to nucleolyticdegradation such as, for example, modified internucleoside linkages(Uhlmann and Psyman, Chemical Reviews 90:543-548, 1990; Schneider andBanner, Tetrahedron Lett 31:335, 1990 which are incorporated byreference), modified nucleic acid bases and/or sugars and the like.

The therapeutic or pharmaceutical compositions of the present inventioncan be administered by any suitable route known in the art including forexample intravenous, subcutaneous, intramuscular, transdermal,intrathecal or intracerebral. Administration can be either rapid as byinjection or over a period of time as by slow infusion or administrationof slow release formulation. For treating tissues in the central nervoussystem, administration can be by injection or infusion into thecerebrospinal fluid (CSF). When it is intended that neurturin orpersephin be administered to cells in the central nervous system,administration can be with one or more agents capable of promotingpenetration of neurturin or persephin across the blood-brain barrier.

Persephin or neurturin can also be linked or conjugated with agents thatprovide desirable pharmaceutical or pharmacodynamic properties. Forexample, persephin or neurturin can be coupled to any substance known inthe art to promote penetration or transport across the blood-brainbarrier such as an antibody to the transferrin receptor, andadministered by intravenous injection. (See for example, Friden et al.,Science 259:373-377, 1993 which is incorporated by reference).Furthermore, persephin or neurturin can be stably linked to a polymersuch as polyethylene glycol to obtain desirable properties ofsolubility, stability, half-life and other pharmaceutically advantageousproperties. (see for example Davis et al. Enzyme Eng 4:169-73, 1978,Burnham, Am J Hosp Pharm 51:210-218, 1994 which are incorporated byreference).

The compositions are usually employed in the form of pharmaceuticalpreparations. Such preparations are made in a manner well known in thepharmaceutical art. One preferred preparation utilizes a vehicle ofphysiological saline solution, but it is contemplated that otherpharmaceutically acceptable carriers such as physiologicalconcentrations of other non-toxic salts, five percent aqueous glucosesolution, sterile water or the like may also be used. It may also bedesirable that a suitable buffer be present in the composition. Suchsolutions can, if desired, be lyophilized and stored in a sterileampoule ready for reconstitution by the addition of sterile water forready injection. The primary solvent can be aqueous or alternativelynon-aqueous. Persephin or neurturin can also be incorporated into asolid or semi-solid biologically compatible matrix which can beimplanted into tissues requiring treatment.

The carrier can also contain other pharmaceutically-acceptableexcipients for modifying or maintaining the pH, osmolarity, viscosity,clarity, color, sterility, stability, rate of dissolution, or odor ofthe formulation. Similarly, the carrier may contain still otherpharmaceutically-acceptable excipients for modifying or maintainingrelease or absorption or penetration across the blood-brain barrier.Such excipients are those substances usually and customarily employed toformulate dosages for parenteral administration in either unit dosage ormulti-dome form or for direct infusion into the cerebrospinal fluid bycontinuous or periodic infusion.

Dose administration can be repeated depending upon the pharmacokineticparameters of the dosage formulation and the route of administrationused.

It is also contemplated that certain formulations containing persephinor neurturin are to be administered orally. Such formulations arepreferably encapsulated and formulated with suitable carriers in soliddosage forms. Some examples of suitable carriers, excipients, anddiluents include lactose, dextrose, sucrose, sorbitol, mannitol,starches, gum acacia, calcium phosphate, alginates, calcium silicate,microcrystalline cellulose, polyvinylpyrrolidone, cellulose, gelatin,syrup, methyl cellulose, methyl- and propylhydroxybenzoates, talc,magnesium, stearate, water, mineral oil, and the like. The formulationscan additionally include lubricating agents, wetting agents, emulsifyingand suspending agents, preserving agents, sweetening agents or flavoringagents. The compositions may be formulated so as to provide rapid,sustained, or delayed release of the active ingredients afteradministration to the patient by employing procedures well known in theart. The formulations can also contain substances that diminishproteolytic degradation and promote absorption such as, for example,surface active agents.

The specific dose is calculated according to the approximate body weightor body surface area of the patient or the volume of body apace to beoccupied. The dose will also be calculated dependent upon the particularroute of administration selected. Further refinement of the calculationsnecessary to determine the appropriate dosage for treatment is routinelymade by those of ordinary skill in the art. Such calculations can bemade without undue experimentation by one skilled in the art in light ofthe activity of neurturin or GDNF. With neurturin the activity in targetcells data is disclosed herein and in copending application Ser. NO.08/519,777, now U.S. Pat. No. 5,739,307 and in the case or persephin,the concentration required for activity at the cellular level isbelieved to be similar to that of neurturin. Persephin activity on aparticular target cell type can be determined by routineexperimentation. Exact dosages are determined in conjunction withstandard dose-response studies. It will be understood that the amount ofthe composition actually administered will be determined by apractitioner, in the light of the relevant circumstances including thecondition or conditions to be treated, the choice of composition to beadministered, the age, weight, and response of the individual patient,the severity of the patient's symptoms, and the chosen route ofadministration.

In one embodiment of this invention, persephin or neurturin may betherapeutically administered by implanting into patients vectors orcells capable of producing a biologically-active form of persephin orneurturin or a precursor of persephin or neurturin, i.e. a molecule thatcan be readily converted to a biological-active form of neurturin by thebody. In one approach cells that secrete persephin or neurturin may beencapsulated into semipermeable membranes for implantation into apatient. The cells can be cells that normally express persephin orneurturin or a precursor thereof or the cells can be transformed toexpress persephin or neurturin or a precursor thereof. It is preferredthat the cell be of human origin and that the persephin or neurturin behuman persephin or neurturin when the patient is human. However, theformulations and methods herein can be used for veterinary as well ashuman applications and the term “patient” as used herein is intended toinclude human and veterinary patients.

Cells can be grown ex vivo for use in transplantation or engraftmentinto patients (Muench et al., Leuk & Lymph 16:1-11, 1994 which isincorporated by reference). In another embodiment of the presentinvention, persephin or neurturin is used to promote the ex vivoexpansion of a cello for transplantation or engraftment. Current methodshave used bioreactor culture systems containing factors such aserythropoietin, colony stimulating factors, stem call factor, andinterleukins to expand hematopoietic progenitor cells for erythrocytes,monocytes, neutrophils, and lymphocytes (Verfaillie, Stem Cells12:466-476, 1994 which is incorporated by reference). These stem cellscan be isolated from the marrow of human donors, from human peripheralblood, or from umbilical cord blood cells. The expanded blood cells areused to treat patients who lack these cells as a result of specificdisease conditions or as a result of high dose chemotherapy fortreatment of malignancy (George, Stem Cells 12( Suppl 1):249-255, 1994which is incorporated by reference). In the case of cell transplantafter chemotherapy, autologous transplants can be performed by removingbone marrow cells before chemotherapy, expanding the cells ex vivo usingmethods that also function to purge malignant cells, and transplantingthe expended cells back into the patient following chemotherapy (forreview see Rummel and Van Zant, J Hematotherapy 3:213-218, 1994 which isincorporated by reference). Since persephin or neurturin Is believed tobe expressed in the developing animal in blood, bone marrow and liver,tissues where proliferation and differentiation of progenitor cellsoccur, it is believed that persephin or neurturin can function toregulate the proliferation of hematopoietic stem cells and thedifferentiation of mature hematopoietic cells. Thus, the addition ofpersephin or neurturin to culture systems used for ex vivo expansion ofcells could stimulate the rate at which certain populations of cellsmultiply or differentiate, and improve the effectiveness of theseexpansion systems in generating cells needed for transplant.

It is also believed that persephin or neurturin can be used for the exvivo expansion of precursor cells in the nervous system. Transplant orengraftment of cells is currently being explored as a therapy fordiseases in which certain populations of neurons are lost due todegeneration such as, for example, in Parkinson's disease (Bjorklund,Curr Opin Neurobiol 2:683-689, 1992 which is incorporated by reference).Neuronal precursor cells can be obtained from animal or human donors orfrom human fetal tissue and then expanded in culture using persephin orneurturin or other growth factors. These cells can then be engraftedinto patients where they would function to replace some of the cellslost due to degeneration. Because neurotrophins have been shown to becapable of stimulating the survival and proliferation of neuronalprecursors cells such as, for example, NT-3 stimulation of sympatheticneuroblast cells (Birren et al., Develop 119:597-610, 1993 which isincorporated by reference), persephin or neurturin could also functionin similar ways during the development of the nervous system and couldbe useful in the ex vivo expansion of neuronal cells.

In a number of circumstance it would be desirable to determine thelevels of persephin or neurturin in a patient. The identification ofpersephin or neurturin along with the present report that persephin andneurturin are expressed by a number of tissues provides the basis forthe conclusion that the presence of persephin or neurturin serves anormal physiologic function related to cell growth and survival. Indeed,other neurotrophic factors are known to play a role in the function ofneuronal and non-neuronal tissues. (For review see Scully and Otten,Cell Biol Int 19:459-469, 1995; Otten and Gadient, Int J DevlNeurosciences 13:147-151, 1995 which are incorporated by reference).Endogenously produced persephin or neurturin may also play a role incertain disease conditions, particularly where there is cellulardegeneration such as in neurodegenerative conditions or diseases. Otherneurotrophic factors are known to change during disease conditions. Forexample, in multiple sclerosis, levels of NGF protein in thecerebrospinal fluid are increased during acute phases of the disease(Bracci-Laudiero et al., Neuroscience Lett 147:9-12, 1992 which isincorporated by reference) and in systemic lupus erythematosus there isa correlation between inflammatory episodes end NGF levels in sera(Bracci-Lauiedro et al. NeuroReport 4:563-565, 1993 which isincorporated by reference).

Given that neurturin is expressed in blood cells, bone marrow and meatcells, and it la believed that persephin is similarly expressed, it islikely that the level of persephin or neurturin may be altered in avariety of conditions and that quantification of persephin or neurturinlevels would provide clinically useful information. Furthermore, in thetreatment of degenerative conditions, compositions containing eitherpersephin or neurturin or both can be administered and it would likelybe desirable to achieve certain target levels of persephin and/orneurturin, as the case may be, in sera, in cerebrospinal fluid or in anydesired tissue compartment. It would, therefore, be advantageous to beable to monitor the levels of the particular growth factor, persephin orneurturin, in a patient. Accordingly, the present invention alsoprovides methods for detecting the presence of persephin or fordetecting the presence of neurturin in a sample from a patient.

The term “detection” as used herein in the context of detecting thepresence of persephin or neurturin in a patient is intended to includethe determining of the amount of persephin or neurturin or the abilityto express an amount of persephin or neurturin in a patient, thedistinguishing of persephin or neurturin from other growth factors, theestimation of prognosis in terms of probable outcome of a degenerativedisease and prospect for recovery, the monitoring of the persephin orneurturin levels over a period of time as a measure of status of thecondition, and the monitoring of persephin or neurturin levels fordetermining a preferred therapeutic regimen for the patient.

To detect the presence of persephin or neurturin in a patient, a sampleis obtained from the patient. The sample can be a tissue biopsy sampleor a sample of blood, plasma, serum, CSF or the like. Neurturin isexpressed in a wide variety of tissues as shown in example 9 and it isbelieved that persephin as well is secreted in a number of tissues.Thus, samples for detecting persephin or neurturin can be taken from anytissues expressing the particular growth factor. When assessingperipheral levels of persephin or neurturin, it is preferred that thesample be a sample of blood, plasma or serum. When assessing the levelsof persephin or neurturin in the central nervous system a preferredsample is a sample obtained from cerebrospinal fluid.

In some instances it is desirable to determine whether the persephin orneurturin gene is intact in the patient or in a tissue or call linewithin the patient. By an intact persephin or neurturin gene it is meantthat there are no alterations in the gene such as point mutations,deletions, insertions, chromosomal breakage, chromosomal rearrangementsand the like wherein such alteration might alter production of persephinor neurturin or alter its biological activity, stability or the like tolead to disease processes or susceptibility to cellular degenerativeconditions. Conversely, by a non-intact persephin or neurturin gene itis meant that such alterations are present. Thus, in one embodiment ofthe present invention a method is provided for detecting andcharacterizing any alterations in the persephin or neurturin gene. Themethod comprises providing an oligonucleotide that contains thepersephin or neurturin cDNA, genomic DNA or a fragment thereof or aderivative thereof. By a derivative of an oligonucleotide, it is meantthat the derived oligonucleotide is substantially the same as thesequence from which it is derived in that the derived sequence hassufficient sequence complementarily to the sequence from which it isderived to hybridize to the persephin or neurturin gene. The derivednucleotide sequence is not necessarily, physically derived from thenucleotide sequence, but may be generated in any manner including forexample, chemical synthesis or DNA replication or reverse transcriptionor transcription.

Typically, patient genomic DNA is isolated from a cell sample from thepatient and digested with one or more restriction endonucleases such as,for example, TaqI and AluI. Using the Southern blot protocol, which iswell known in the art, this assay determines whether a patient or aparticular tissue in a patient has an intact persephin or neurturin geneor a persephin or neurturin gene abnormality.

Hybridization to the persephin or neurturin gene would involvedenaturing the chromosomal DNA to obtain a single-stranded DNA;contacting the single-stranded DNA with a gene probe associated with thepersephin or neurturin gene sequence; and identifying the hybridizedDNA-probe to detect chromosomal DNA containing at least a portion of thehuman persephin or neurturin gene.

The term “probe” as used herein refers to a structure comprised of apolynucleotide which forms a hybrid structure with a target sequence,due to complementarity of probe sequence with a sequence in the targetregion. Oligomers suitable for use as probes may contain a minimum ofabout 8-12 contiguous nucleotides which are complementary to thetargeted sequence and preferably a minimum of about 20.

The persephin or neurturin gene probes of the present invention can beDNA or RNA oligonucleotides end can be made by any method known in theart such as, for example, excision, transcription or chemical synthesis.Probes may be labeled with any detectable label known in the art suchas, for example, radioactive or fluorescent labels or enzymatic marker.Labeling of the probe can be accomplished by any method known in the artsuch as by PCR, random priming, and labelling, nick translation or thelike. One skilled in the art will also recognize that other methods notemploying a labeled probe can be used to determine the hybridization.Examples of methods that can be used for detecting hybridization includeSouthern blotting, fluorescence in situ hybridization, and single-strandconformation polymorphism with PCR amplification.

Hybridization is typically carried out at 25-45° C., more preferably at32-40° C. and more preferably et 37-38° C. The time required forhybridization is from about 0.25 to about 96 hours, more preferably fromabout one to about 72 hours, and most preferably from about 4 to about24 hours.

Persephin or neurturin gene abnormalities can also be detected by usingthe PCR method and primers that flank or lie within the persephin orneurturin gene. The PCR method is well known in the art. Briefly, thismethod is performed using two oligonucleotide primers which are capableof hybridizing to the nucleic acid sequences flanking a target sequencethat lies within a persephin or neurturin gene and amplifying the targetsequence. The terms “oligonucleotide primer” as used herein refers to ashort strand of DNA or RNA ranging in length from about 8 to about 30bases. The upstream and downstream primers are typically from about 20to about 30 base pairs in length and hybridize to the flanking regionsfor replication of the nucleotide sequence. The polymerization iscatalyzed by a DNA-polymerase in the presence of deoxynucleotidetriphosphates or nucleotide analogs to produce double-stranded DNAmolecules. The double strands are then separated by any denaturingmethod including physical, chemical or enzymatic. Commonly, the methodof physical denaturation is used involving heating the nucleic acid,typically to temperatures from about 80° C. to 105° C. for times rangingfrom about 1 to about 10 minutes. The process is repeated for thedesired number of cycles.

The primers are selected to be substantially complementary to the strandof DNA being amplified. Therefore, the primers need not reflect theexact sequence of the template, but must be sufficiently complementaryto selectively hybridize with the strand being amplified.

After PCR amplification, the DNA sequence comprising persephin orneurturin or pre-pro persephin or neurturin or a fragment thereof isthen directly sequenced and analyzed by comparison of the sequence withthe sequences disclosed herein to identify alterations which mightchange activity or expression levels or the like.

In another embodiment a method for detecting persephin or neurturin isprovided based upon an analysis of tissue expressing the persephin geneor the neurturin gene. Certain tissues such as those identified below inexample 9 have been found to express the neurturin gene. It is alsobelieved that a number of tissues will express the persephin gene basedupon the observations for neurturin and the identification herein ofbrain and heart as tissues expressing persephin. The method compriseshybridizing a polynucleotide to mRNA from a sample of tissues thatnormally express the persephin gene or the neurturin gene. The sample isobtained from a patient suspected of having an abnormality in thepersephin gene or the neurturin gene or in the persephin gene or theneurturin gene of particular cells. In the case of neurturin, thepolynucleotide comprise SEQ ID NO:11 or a derivative thereof or afragment thereof. In the case of persephin, the polynucleotide comprisesSEQ ID NO:105 or SEQ ID NO:107 or the human ortholog of persephin orderivatives thereof or fragments thereof.

To detect the presence of mRNA encoding persephin protein or neurturinprotein, a sample is obtained from a patient. The sample can be fromblood or from a tissue biopsy sample. The sample may be treated toextract the nucleic acids contained therein. The resulting nucleic acidfrom the sample is subjected to gel electrophoresis or other sizeseparation techniques.

The mRNA of the sample is contacted with a DNA sequence serving as aprobe to form hybrid duplexes. The use of a labeled probes as discussedabove allows detection of the resulting duplex.

When using the cDNA encoding persephin protein or neurturin protein or aderivative of the cDNA as a probe, high stringency conditions can beused in order to prevent false positives, that is the hybridization andapparent detection of persephin or neurturin nucelotide sequences whenin fact an intact and functioning persephin gene or neurturin gene isnot present. When using sequences derived from the persephin orneurturin cDNA, less stringent conditions could be used, however, thiswould be a less preferred approach because of the likelihood of falsepositives. The stringency of hybridization is determined by a number offactors during hybridization and during the washing procedure, includingtemperature, ionic strength, length of time and concentration offormamide. These factors are outlined in, for example, Sambrook et al.(Sambrook, et al., 1989, supra).

In order to increase the sensitivity of the detection in a sample ofmRNA encoding the persephin protein or neurturin protein, the techniqueof reverse transcription/polymerization chain reaction (RT/PCR) can beused to amplify cDNA transcribed from mRNA encoding the persephinprotein or the neurturin protein. The method of RT/PCR is well known inthe art (see example 9 and FIG. 6 below).

The RT/PCR method can be performed as follows. Total cellular RNA isisolated by, for example, the standard guanidium isothiocyanate methodand the total RNA is reverse transcribed. The reverse transcriptionmethod involves synthesis of DNA on a template of RNA using a reversetranscriptase enzyme and a 3′ end primer. Typically, the primer containsan oligo(dT) sequence. The cDNA thus produced is then amplified usingthe PCR method and persephin specific primers or neurturin specificprimers. (Belyavsky et al, Nucl Acid Res 17:2919-2932, 1989; Krug andBerger, Methods in Enzymology, Academic Press, N.Y., Vol. 152, pp.316-325, 1987 which are incorporated by reference).

The polymerase chain reaction method is performed as described aboveusing two oligonucleotide primers that are substantially complementaryto the two flanking regions of the DNA segement to be amplified.

Following amplification, the PCR product is then electrophoresed anddetected by ethidium bromide staining or by phosphoimaging.

The present invention further provides for methods to detect thepresence of the persephin protein or the neurturin protein in a sampleobtained from a patient. Any method known in the art for detectingproteins can be used. Such methods include, but are not limited toimmunodiffusion, immunoelectrophoresis, immunochemical methods,binder-ligand assays, immunohistochemical techinques, agglutination andcomplement assays. (for example see Basic and Clinical Immunology, Sitesand Terr, eds., Appleton & Lange, Norwalk, Conn. pp 217-262, 1991 whichis incorporated by reference). Preferred are binder-ligand immunoassaymethods including reacting antibodies with an epitope or epitopes of thepersephin protein or reacting antibodies with an epitope or epitopes ofthe neurturin protein and competitively displacing a labeled persephinprotein or a labeled neuturin protein or derivative thereof.

As used herein, a derivative of the persephin protein or a derivative ofthe neurturin protein is intended to include a polypeptide in whichcertain amino acids have been deleted or replaced or changed to modifiedor unusual amino acids wherein the persephin derivative or the neurturinderivative is biologically equivalent to persephin or neurturin,respectively, and wherein the polypeptide derivative cross-reacts withantibodies raised against the persephin protein or the neurturinprotein, respectively. By cross-section it is meant that an antibodyreacts with an antigen other than the one that induced its formation.

Numerous competitive and non-competitive protein binding immunoassaysare well known in the art. Antibodies employed in such assays may beunlabeled, for example as used in agglutination tests, or labeled foruse in a wide variety of assays methods. Labels that can be used includeradionuclides, enzymes, fluorescers, chemiluminescers, enzyme substratesor co-factors, enzyme inhibitors, particles, dyes and the like for usein radioimmunoassay (RIA), enzymes immunoassays, e.g., enzyme-linkedimmunosorbent assay (ELISA), fluorescent immunoassays and the like.

Polyclonal or monoclonal antibodies to the persephin protein and to theneurturin protein or an epitope thereof can be made for use inimmunoassays by any of a number of methods known in the art. By epitopereference is made to an antigenic determinant of a polypeptide. Anepitope could comprise 3 amino acids in a spacial conformation which isunique to the epitope. Generally an epitope consists of at least 5 suchamino acids. Methods of determining the spatial conformation of aminoacids are known in the art, and include, for example, x-raycrystallography and 2 dimensional nuclear magnetic resonance.

One approach for preparing antibodies to a protein is the selection andpreparation of an amino acid sequence of all or part of the protein,chemically synthesizing the sequence and injecting it into anappropriate animal, usually a rabbit or a mouse (See Example 10).

Oligopeptides can be selected as candidates for the production of anantibody to the presephin protein or for the production of an antibodyto the neurturin protein based upon the oligopeptides lying inhydrophilic regions, which are thus likely to be exposed in the matureprotein.

Antibodies to persephin or to neurturin can also be raised againstoligopeptides that include one or more of the conserved regionsidentified herein such that the antibody can cross-react with otherfamily members. Such antibodies can be used to identify and isolate theother family members.

Methods for preparation of the persephin protein or to the neurturinprotein or an epitope thereof include, but are not limited to chemicalsynthesis, recombinant DNA techniques or isolation from biologicalsamples. Chemical synthesis of a peptide can be performed, for example,by the classical Merrifeld methof of solid phase peptide synthesis(Merrifeld, J Am Chem Soc 85:2149, 1963 which is incorporated byreference) or the FMOC strategy on a Rapdi Automated Multiple PeptideSynthesis system (DuPont Company, Wilmington, Del.) (Caprino and Han, JOrg Chem 37:3404, 1972 which is incorporated by reference).

Polyclonal antibodies can be prepared by immunizing rabbits or otheranimals by injecting antigen followed by subsequent boosts atappropriate intervals. The animals are bled and sera assayed againstpurified persephin protein or purified neurturin protein usually byELISA or by bioassay based upon the ability to block the action ofpersephin or neurturin, as the case may be. When using avian species,e.g. chick, turkey and the like, the antibody can be isolated from theyolk of the egg. Monoclonal antibodies can be prepared after the methodof Milstein and Kohler by fusing splenocytes from immunized mice withcontinuously replicating tumor cells such as myeloma or lymphoma cells.(Milstein and Kohler Nature 256:495-497, 1975; Gulfre and Milstein,Methods in Enzymology: Immunochemical Techniques 73:1-46, Langone andBanatis eds., Academic Press, 1981 which are incorporated by reference).The hybridoma cells so formed are then cloned by limiting dilutionmethods and supernates assayed for antibody production by ELISA, RIA orbioassay.

The unique ability of antibodies to recognize and specifically bind totarget proteins provides an approach for treating an over expression ofthe protein. Thus, another aspect of the present invention provides fora method for preventing or treating diseases involving over expressionof the persephin protein or the neurturin protein by treatment of apatient with specific antibodies to the persephin protein or to theneurturin protein, respectively.

Specific antibodies, either polyclonal or monoclonal, to the persephinprotein or to the neurturin protein can be produced by the suitablemethod known in the art as discussed above. For example, murine or humanmonoclonal antibodies can be produced by hybridoma technology or,alternatively, the persephin protein or the neurturin protein, or animmunologically active fragment thereof, or an anti-idiotypic antibody,or fragment thereof can be administered to an animal to elicit theproduction of antibodies capable of recognizing and binding to thepersephin protein or to the neurturin protein. Such antibodies can befrom any class of antibodies including, but not limited to IgG, IgA,IgM, IgD, and IgE or in the case of avian species, IgY and from anysubclass of antibodies.

Preferred embodiments of the invention are described in the followingexamples. Other embodiments within the scope of the claims herein willbe apparent to one skilled in the art from consideration of thespecification or practice of the invention as disclosed herein. It isintended that the specification, together with the examples, beconsidered exemplary only, with the scope and spirit of the inventionbeing indicated by the claims which follow the examples.

EXAMPLE 1

This example illustrates the isolation and purification of neurturinfrom CHO cell conditioned medium.

Preparation of CHO cell conditioned medium:

A derivative of DG44 Chinese hamster ovary cells, DG44CHO-pHSP-NGFI-B(CHO) cells, was used (Day et al, J Biol Chem 265:15253-15260, 1990which is incorporated by reference). As noted above, the inventors havealso obtained neurturin in partially purified form from otherderivatives of DG44 Chinese hamster ovary cells. The CHO cells weremaintained in 20 ml medium containing minimum essential medium (MEM)alpha (Gibco-BRL No. 12561, Gaithersburg, Md.) containing 10% fetal calfserum (Hyclone Laboratories, Logan, Utah) 2 mM 1-glutamine, 100 U/mlpenicillin, 100 μg/ml streptomycin and 25 nM methotrexate using 150 cm²flasks (Corning Inc., Corning N.Y.). For passage and expansion, mediumfrom a confluent flask was aspirated; the cells were washed with 10 mlphosphate buffered saline (PBS) containing in g/l, 0.144 KH₂PO₄, 0.795Na₂HPO₄ and 9.00 NaCl; and the flask was then incubated for 2-3 minuteswith 2 ml 0.25% trypsin in PBS. Cells were then knocked off the flasksurface, 8 ml of medium were added and cells were triturated severaltimes with a pipette. The cells were split 1:5 or 1:10, incubated at 37°C. under an atmosphere of 5% CO₂ in air and grown to confluence for 3-4days.

The cell culture was then expanded into 850 cm² roller bottles (BectonDickinson, Bedford, Mass.). A confluent 150 cm² flask was trypsinizedand seeded into one roller bottle containing 240 ml of the abovemodified MEM medium without methotrexate. The pH was maintained eitherby blanketing the medium with 5% CO₂ in air or by preparing the mediumwith 25 mM HEPES pH 7.4 (Sigma, St. Louis, Mo.). The roller bottles wererotated at 0.8-1.0 revolutions per minute. Cells reached confluence in 4days.

For collecting conditioned medium, serum-free CHO cell (SF-CHO) mediumwas used. SF-CHO was prepared using 1:1 DME/F12 base medium, which wasprepared by mixing 1:1 (v/v) DMEM (Gibco-BRL product No. 11965,Gibco-BRL, Gaithersburg, Md.) with Ham's F12 (Gibco-BRL product No.11765). The final SF-CHO medium contained 15 mM HEPES pH 7.4 (Sigma, St.Louis, Mo.), 0.5 mg/ml bovine serum albumin (BSA, Sigma, St. Louis,Mo.), 25 μg/ml heparin, (Sigma, St. Louis, Mo.), 1Xinsulin-transferrin-selenite supplement (bovine insulin, 5 μg/ml; humantransferrin, 5 μg/ml; sodium selentine, 5 ng/ml; Sigma, St. Louis, Mo.),2 mM 1-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin. Themedium from the confluent roller bottles was removed and the cellswashed once with 30 ml SF-CHO medium to remove serum proteins. Cellswere then incubated at 37° C. for 16-24 hrs in 80 ml SF-CHO medium tofurther remove serum proteins. The 80 ml medium was removed anddiscarded. A volume of 120 ml of SF-CHO medium was added to the flaskand the cells incubated at 37° C. Every 48 hrs thereafter, 120 ml wascollected and replaced with the same volume of SF-CHO medium.

Collected media was pooled and centrifuged at 4° C. in polypropyleneconical tubes to remove cellular debris and the supernatant stored at−70° C. Media was collected 5 times over 10 days to yield a total ofapproximately 600 ml conditioned medium per roller bottle.

Fractions collected from the columns at each stage of purification wereassayed for biological activity using the neuronal survival assay andfor protein content by the dye binding assay of Bradford (Anal Biochem72:248 et seq., 1976 which is incorporated by reference). The total mgof protein in the starting volume, typically 50 liters, of conditionedmedium was determined.

Superior Cervical Ganglion Survival Assay:

The neurotrophic activity of CHO conditioned medium starting materialand at various stages of purification was assessed using the superiorcervical ganglion survival assay system previously reported (Martin, etal J of Cell Biology 106:829-844; Deckwerth and Johnson, J Cell Bio123:1207-1222, 1993 which are incorporated by reference). Primarycultures of sympathetic neurons from superior cervical ganglion (SCG)were prepared by dissecting tissue from Day 20-21 rat embryo (E20-E21).The SCG's were placed in Leibovitz's L15 with 1-glutamine medium (Cat#11415-023 Gibco-BRL, Gaithersburg, Md.), digested for 30 minutes with 1mg/ml collagenase (Cat #4188 Worthington Biochemica., Freehold, N.J.) inLeibovitz's L15 medium at 37° C., followed by a 30 minute digestion intrypsin-lyophilized & irradiated (Type TRLVMF Cat #4454 WorthingtonBiochemical, Freehold, N.J.) which was resuspended in modified Hanks'Balanced Salt Solution (Cat #H-8389 Sigma Chemical Co., St. Louis Mo.).The digestion was stopped using AM50 which contains Minimum EssentialMedium with Earle's salts and without 1-glutamine (Cat #11090-016Gibco-BRL), 10% fetal calf serum (Cat #1115 Hyclone Laboratories, Logan,Utah), 2 mM 1-glutamine (Cat #G5763 Sigma Chemical Co., St. Louis, Mo.),20 μM FuDr (F-0503 Sigma Chemical Co., St. Louis, Mo.), 20 μM Uridine(Cat #3003 Sigma Chemical Co., St. Louis, Mo.), 100 U/ml penicillin, 100μg/ml Streptomycin, and 50 ng/ml 2.5 S NGF. The cells were dissociatedinto a suspension of single cells using a silanized and flame-polishedPasteur pipet. After filtration of the suspension through a nitex filter(size 3-20/14, Tetko Inc., Elmsford, N.Y.), the cells were placed inAM50 medium as above and preplated on a 100 mm Falcon or Primariaculture dish (Becton Dickinson Labware, Lincoln Park, N.J.) to reducethe number of non-neuronal cells. After 2 hours, the medium containingthe unattached neuronal cells was removed from these dishes andtriturated again through a silanized and flame-polished Pasteur pipet.The single cell suspension was plated on 24-well tissue culture plates(Costar, Wilmington, Mass.) that have been previously coated with adouble layer of collagen, one layer of collagen that had been ammoniatedand a second layer of collagen that had been air dried. They wereallowed to attach for 30 minutes to 2 hours. A specific number of viablecells, usually about 1200 to about 3000 total cells per well, or aspecific percentage of the ganglion, usually 25% of the cells obtainedper ganglion were placed into each well. When cell counts were to beperformed they were placed in the 24-well dishes as stated above oralternatively, on 2-well chamber slides (Nunc, Naperville, Ill.).Cultures were then incubated for 5-6 days at 37° in AM50 medium in a 5%CO₂/95% air atmosphere. The death of the cultured neurons was induced byexchanging the medium with medium without NGF and with 0.05% goatanti-NGF (final titer in the wells is 1:10). This NGF-deprivationresults in death of the neurons over a period of 24-72 hours. Aliquotsof partially purified or purified factor, or appropriate controls, wereadded to the cultures at the time of NGF removal to determine theability to prevent the neuronal death.

Evaluation of the ability of column fraction, gel eluates, or purifiedfactor to prevent neuronal death was by visual inspection of culturesunder phase contrast microscopy. Viable neurons remained phase brightwith intact neurities, whereas dead neurons were shrunken, phase dark,had irregular membranes and neurites were fragmented (FIG. 3). Whereprecise quantitation of neuronal survival was required, the cultureswere fixed in 4% paraformaldehyde or 10% Formalin in PBS, and stainedwith crystal violet solution, (Huntoon Formula Harleco E.M. DiagnosticsSystems, Gibbstown, N.J.). When using 24 well dishes, 1 μl crystalviolet solution was added to each well containing 10% formalin and thecells were counted using a phase contrast microscope. If the 2-wellchamber slides were used, the cultures were fixed, stained with crystalviolet, destained with water, dehydrated in increasing ethanolconcentrations to toluene, and mounted in a toluene-based mountingsolution. Neurons were scored as viable if they had a clear nucleolusand nuclei and were clearly stained with crystal violet.

The neuronal death at 72 hours in shown in FIG. 3B. Also shown are (A)the positive control cells maintained with nerve growth factor and (C)the cells treated with anti-NGF and neurturin (approximately 3 ng/ml)showing survival of neurons.

Activity was quantitated by calculation of a “survival unit”. The totalsurvival units in a sample were defined as the minimal volume of analiquot of the sample which produced maximal survival divided into thetotal volume of that sample. Specific activity was calculated as thesurvival units divided by the mg total protein.

Survival units were determined in an assay using approximately 1200viable neurons in a 0.5 ml culture assay and a culture period of 48hours following addition of the fraction. Survival was assessed visuallyafter the 48 hours. Intrinsic activity as shown in FIG. 4 was determinedin an assay using approximately 2700 neurons and a culture period of 72hours. Survival was assessed by fixing the neurons and counting thenumber of surviving neurons. Because the stability, as assessed byhalf-life of activity, for neurturin decreases as the number of neuronsincreases, the intrinsic activity measurement would be expected to belower than that predicted by Specific Activity determinations. Theintrinsic activity measurement would also be expected to be lower thanthat predicted by specific activity because the survival was measuredafter 72 hours instead of 48 hours.

To ensure the reproducibility of these activity unit assays, it wasnecessary to plate the primary neuronal cultures at reproducible celldensities, as the stability of the activity decreases significantly withincreasing neuronal density. The range of cell densities was from about1200 to about 2700 cells per well. The presence of soluble heparin inthe assay medium had no effect on the short-term (˜3 days) stability ofthe survival activity.

Purification of Neurturin:

Pooled conditioned medium was filtered through 0.2 μl pore bottle-topfilters (cellulose acetate membrane, Corning Inc., Corning, N.Y.).Typically 50 liters of conditioned medium was used and processed in 25liter batches. Each 25 liter batch was introduced at a rate of 20 ml/minonto a 5×5 cm column containing 100 ml heparin-agarose (Sigma, St. Lous,Mo.) equilibrated with 25 μmM HEPES, pH 7.4 buffer with 150 mM NaCl. thecolumn was then washed with approximately 1000 ml 25 mM HEPES, pH 7.4buffer containing 0.5 M NaCl at 20 ml/min and the activity was theneluted with 25 mM HEPES, pH 7.4 buffer containing 1.0 M NaCl. Afterswitching to the 1.0M NaCL elution buffer, the first 50 ml of buffer wasdiscarded and, thereafter, one 300 ml fraction was collected.

Pooled material eluted from the Heparin-agarose column was then diluted1:1 (v/v) with 25 mM HEPES, pH 7.4 buffer containing 0.04% TWEEN 20 to aNaCl concentration of 0.5 M and introduced into a 1:5 cm×9 cm columncontaining 16 ml SP SEPHAROSE® High Performance ion exchange resin(Pharmacia, Piscataway, N.J.) equilibrated in 25 mM HEPES 7.4 containing0.5 M NaCl and 0.02% TWEEN 20. The column was then washed with 160 ml 25mM HEPES, pH 7.4 buffer containing 0.5 M NaCl and 0.02% TWEEN 20 and theactivity was eluted with 25 mM HEPES, pH 7.4 buffer containing 1.0 MNaCl and 0.02% TWEEN 20 at a flow rate of 2 ml/min. One 50 ml fractionwas collected after the first 7 ml of eluate from the column.

Material eluted from the SP SEPHAROSE® column was fractionated usingfast protein liquid chromatography (FPLC) on a Chelating Superose HR10/2 column charged with Cu⁺⁺ (Pharmacia, Piscataway, N.J.). the columnhad been prepared by washing with 10 ml water, charging with 3 ml of 2.5mg/ml CuSO₄·5H₂O, washing with 10 ml water, and equilibrating with 10 mlof 25 mM HEPES pH 7.4 buffer containing 1.0 M NaCl and 0.02% TWEEN 20.The eluate was introduced into the column in 25 mM HEPES, pH 7.4 buffercontaining 1.0 M NaCl at a rate of 1.0 ml/min. The bound proteins wereeluted with a linear gradient of increasing glycine concentration (0-300mM) in 25 mM HEPES, pH 7.4 buffer containing 1.0 M NaCl at a rate of 1.0ml/min. The gradient was produced by a Pharmacia FPLC system using anLCC-500 controller and P-500 pumps to establish a 0-300 mM glycinegradient in 40 ml at 1.0 ml/min, thus increasing the gradient by 7.5 mMglucine per min. One ml fractions were collected and assayed for SCGsurvival promotion. Peak activity was observed in fractions 17-20, i.e.17-20 min or ml from the start of the gradient.

Absorbance measurement at 280 nM by an in-line UV monitor indicated thatmost proteins eluted prior to the survival activity in fractions 17-20.Thus, significant purification was achieved at this step. A 25 kD bandco-purified with the survival activity.

The combined eluted fractions from the Cu⁺⁺ superose column were dilutedto 0.45 M NaCl using 25 mM HEPES pH 7.4 buffer containing 0.02% TWEEN 20and introduced into a Mono S HR 5/5 cation exchange column (Pharmacia,Piscataway, N.J.) for further FPLC purification. the column had beenequilibrated with 25 mM HEPES pH 7.4 buffer containing 0.45 M NaClcontaining 0.02% TWEEN 20. Bound proteins were eluted with a lineargradient of increasing NaCl concentration (0.45-1.0 M). The gradient wasproduced as described above from 0.45 M-1.0 M NaCl in 35 mls at 1.0ml/min, thus increasing conccentration at 0.0157 M per ml or min.Thirteen 1.0 ml fractions (fractions 1-13) were collected followed by 440.5 ml fractions (fractions 14-53). Peak activity in SCG assay was infractions 26-29. Each fraction was assayed in the SCG survival assayover a range of volumes of from 1.0 to 1.0 μl per 0.5 ml culture medium.

One percent (5 μl) of each fraction was loaded onto a non-reducing, 14%SDS polyacrylamide gel and electrophoresed for 750 V-hr at 25° C.Proteins were visualized by silver stain. The results are shown in FIG.2. Markers shown in lane M on the gel represent 20 ng of Bovine serumalbumin, carbonic anhydrase, B-lactoglobulin, and lysozyme in the orderof descending molecular weight.

A 25 kD band appeared in fractions 25-30, a 28 kD protein elutes earlierin the gradient and an 18 kD elutes later in the gradient. FIG. 2illustrates the survival activity in each of the fractions. The survivalactivity is noted to correspond with the presence and apparent intensityof the 25 kD protein in fractions 25-30.

To demonstrate that the 25 kD band was responsible for survivalpromoting activity, the 25 kD protein was eluted from the polyacrylamidegel after electrophoresis and assayed for survival activity in the SCGassay. After electrophoresis of 150 μl of the SP SEPHAROSE® 1.0 M NaClfraction in one lane of a non-reducing 14% SDS-polyacrylamide gel asabove, the lane was cut into 12 slices and each slice was crushed andeluted by diffusion with rocking in buffer containing 25 mM HEPES, pH7.4, 0.5 M NaCl, 0.02% Tween-20 for 18 hr at 25° C. BSA was added to theeluate to a final concentration of 200 μg/ml and the eluate was filteredthrough a 0.45 micron filter to remove acrylamide gel fragments. Thefiltrate was then added to a SP SEPHAROSE® column to concentrate andpurify the sample. Before eluting the sample, the column was washed oncein 400 μl 25 mM HEPES, pH 7.4 buffer containing 0.5 M NaCl, 0.02%Tween-20 and 200 μg BSA per ml and once in 400 μl 25 mM HEPES, pH 7.4buffer containing 0.02% Tween-20 and 200 μg BSA per ml. The column wasthen washed again in 400 μl of 25 mM HEPES, pH 7.4 buffer containing 0.5M NaCl, 0.02% TWEEN 20 and 200 ug BSA per ml. The sample was eluted with25 mM HEPES, pH 7.4 buffer containing 1.0 M NaCl, 0.02% Tween-20 and 200μg BSA per ml. Samples were then analyzed for survival activity. Onlythe slice corresponding to the 25 kD band showed evidence of survivalactivity. The 25 kD protein purified from CHO cell conditioned media isbelieved to be a homodimer.

The yield from the purification above was typically 1-1.5 μg from 50liters of CHO cell conditioned medium. Overall recovery is estimated tobe 10-30%, resulting in a purification of approximately 390,000 fold.

EXAMPLE 2

This example illustrates the characterization of neurturin and severalmembers of the TGF-β family of growth factors in the SCG assay and thelack of cross reactivity of anti-GDNF antibodies with neurturin.

The SCG assay of the purified protein indicated that the factor ismaximally active at a concentration of approximately 3 ng/ml orapproximately 100 pM and the EC₅₀ was approximately 1.5 ng/ml orapproximately 50 pM in the expected range for a diffusible peptidegrowth factor (FIG. 4).

Several members of the TGF-β family influence neuropeptide geneexpression in sympathetic neurons, while others promote survival ofdifferent neuronal populations. Neuturin, which is a distant member ofthis family of proteins, is capable of promoting virtually completesurvival of sympathetic neurons for 3 days. In addition, furtherculturing of the SCG cells revealed that neurturin could continue tomaintain these neurons for at least 10 days after withdrawal of NGF.

We tested several other members of the TGF-β family for their ability topromote survival in the SCG assay including TGFβ1, activin, BMP-2,BMP-4, BMP-6 and GDNF. Of these factors, only GDNF had survivalpromoting activity, however, the activity of GDNF was much less potentthan neurturin in this activity showing an EC₅₀ of 2-4 nM in the 3-daysurvival assay. The GDNF tested in this assay was rhGDNF produced in E.Coli obtained from Prepro Tech, Inc., Rocky Hill, N.J. The duration ofaction of GDNF was also less than that of neurturin inasmuch as theability of GDNF (50 ng/ml) to maintain survival longer than 3 days wassubstantially diminished. These experiments suggest the possibility thatGDNF is a weak agonist for the neurturin receptor. Furthermore, theinability of activin and BMP-2 to promote survival, in contrast to theirstrong induction of transmitter-related gene expression in these neurons(Fann and Paterson, Int J Dev Neurosci 13:317-330, 1995; Fann andPatterson, J Neurochem 61:1349-1355, 1993) suggests that they signalthrough alternate receptors or signal transduction pathways.

To determine the cross-reactivity of anti-GDNF antibodies with partiallypurified neurturin, SCG neurons, that had been dissected and plated asdescribed in Example 1 were treated on Day 6 with 1 ng/ml, 3 ng/ml, 10ng/ml, or 30 ng/ml GDNF (Prepro Tech, Inc, Rocky Hill, N.J.) in thepresence of anti-NGF alone, or in the presence of anti-NGF and anti-GDNF(goat IgG antibody to E. coli-derived rhGDNF, R & D Systems,Minneapolis, Minn.). A partially purified 1.0 M SP Sepharose fraction ofneurturin was used in the assay at the approximate concentrations of 375pg/ml, 750 pg/ml, 1.5 ng/ml and 3 ng/ml. This fraction was tested in thepresence of anti-NGF alone, and in the presence of anti-NGF andanti-GDNF. The anti-GDNF antibody blocked the survival promotingactivity of GDNFat a concentration up to 30 ng/ml, but did not block thesurvival promoting activity of neurturin.

EXAMPLE 3

This example illustrates the effect of neurturin on sensory neurons in anodose ganglion survival assay.

CHO cell conditioned media that had been partially purified on the SPSepharose column was assayed for neurotrophic activity on sensoryneurons using nodose ganglia. The survival assay is a modification ofthat previously reported above for superior cervical ganlia. Primarydissociated cultures of nodose ganglia were prepared by dissectingtissue from E18 Sprague Dawley rat pups. The nodose ganglia were placedin Leibovitz's L15 with 2 mM 1-glutamine (Cat#11415-023, GIBCO-BRL.Gaithersburg, Md.) as the tissues was dissected, digested for 30 minwith 1 mg/ml collagenase (Cat#4188, Worthington Biochemical, Freehold,N.J.) in Leibovitz's L15 medium at 37° C., followed by 30 min digestionin trypsin (lyophilized and irradiated, type TRLVMF, Cat#4454Worthington Biochemical, Freehold, N.J.), and resuspension to a finalconcentration of 0.25% in modified Hank's Balanced Salt Solution(Cat#H8389, Sigma Chemical Co., St. Louis, Mo.). The digestion wasstopped using AMO-BDNF100, a medium containing Minimum Essential Mediumwith Earle's salts and without 1-glutamine (#11090-016 GIBCO-BRL), 10%fetal Calf Serum (Cat#1115, Hyclone Laboratories, Logan, Utah), 2 mM1-glutamine (Cat#G5763 Sigma Chemical Co., St. Louis, Mo.), 20 μM FuDr(F-0503, Sigma Chemical Co.,), 20 μM Uridine (Cat #3003, Sigma ChemicalCo., St. Louis, Mo.) 100 U/ml penicillin, 100 μg/ml Streptomycin, and100 ng Brain Derived Neurotropic Factor (BDNF, Amgen, Thousand Oaks,Calif.). The cells were dissociated into a suspension of sngle cellsusing a silanized and flame-polished Pasteur pipet in the AMO-BDNF100medium, and preplated on a 100 mm Falcon or Primaria culture dish(Becton Dickinson Labware, Lincoln Park, N.J.) to remove non-neuronalcells. After 2 hours, the medium containing the unattached neuronalcells was removed from these dishes and triturated again through asilanized and flame-polished Pasteur pipet. The single cell suspensionwas plated on 24-well tissue culture plates (Coster, Wilmington, Mass.)that have been previously coated with a double layer of collagen, onelayer of which had been ammoniated and a second layer that had been airdried. Ganglia from ten E18 rat embryos were dissociated into 2.5 mls ofmedia and 100 μl of this suspension was added to each well. The cellswere allowed to attach for 30 min in a 37° C. incubator with 5% CO2/95%air. The wells were fed with AMO-BDNF100 media overnight.

The next day the cells were washed 3 times for 20 min each time with AMOmedium containing no BDNF. The wells were fed with 0.5 ml of this mediaalone or this media containing either 50 ng/ml NGF, 100 ng/ml BDNF(Amgen, Thousand Oaks, Calif.), 100 ng/ml GDNF (Prepro Tech, Inc., RockyHill, N.J.) or 3 ng/ml Neurturin. The cells were incubated at 37° C. ina 5% CO₂/95% air incubator for 3 days, fixed with 10% formalin, stainedwith crystal violet (1 μl/ml 10% formalin) and counted. Survival wasascertained as noted previously.

The neuronal Death at 72 hours is shown in FIG. 10. Neuronal survival ofnodose neurons cultured in BDNF has been previously reported (Thaler etal, Develop Biol 161:338-344, 1994 which is incorporated by reference).This was used as the standard for survival for these neurons and giventhe value of 100% survival. Nodose ganglia that had no trophic support(AMO) showed 20%-30% survival, as did neurons that were cultured in thepresence of 50 ng/ml NGF. Neurons cultured in the presence of 3 ng/mlneurturin and absence of BDNF showed survival similar to those neuronscultured in the presence of BDNF (100 ng/ml). GDNF at a concentration of100 ng/ml promoted greater survival of nodose neurons than did BDNF (100ng/ml). Similar findings with GDNF were recently reported for sensoryneurons from chicken (Ebendal, T. et al, J Neurosci Res 40:276-284 1995which is incorporated by reference).

EXAMPLE 4

This example illustrates the determination of partial amino acidsequences of neurturin isolated from CHO cell conditioned medium.

To obtain N-terminal amino acid sequence from a purified preparation ofapproximately 1 μg of neurturin, the Mono S fractions 26-29 containingthe peak of activity were concentrated to 25 μl by centrifugeultrafiltration in a microcon-3 concentrators (Amicon, Inc., Beverley,Mass.) and loaded onto a non-reducing 14% SDS polyacrylamide gel. Afterelectrophoretic separation, proteins were electroblotted to a PVDFmembrane (Bio-Rad, Hercules, Calif.) and stained with 0.1% CoomassieBlue. The 25 kD band was excised and inserted into the reactioncartridge of an automated sequencer (Model 476, Applied Biosystems(Foster City, Calif.). Phenylthiohydantoin-amino acid (PTH-aa) recoveryin the first 2-3 cycles of automated sequencing by Edman degradationindicated a sequencing yield of 4 pmoles, which which was approximately10% of the estimated amount of protein loaded on the SDS gel.

Two N-terminal sequencing runs were performed from two 50 literpurification preparations. In the first run, 1 μg of protein in 3 pooledfractions of 1.5 ml total volume were concentrated to 25 μl andelectroblotted at 100V for 2 hrs at 25° C. using an electroblot bufferof 10 mM CAPS pH 11.0 buffer (Sigma, St. Louis, Mo.) containing 5%methanol. The maino acid sequence was obtained from 13 cycles of Edmandegradation and the sequencing yield was 4 pmoles as above.

In the second run, 1.5 μg of protein in 4 pooled fractions of 2.0 mltotal volume were concentrated to 25 μl and electroblotted at 36V for 12hours at 4° C. using an electroblot buffer of 25 mM Tris, 192 mMglycine, 0.04% SDS and 17% MeOH. Sequencing yield was 15 pmoles and thesequence after 16 cycles was SGARPXGLRELEVSVS (SEQ ID NO:3). Thesequence obtained after 16 cycles corresponded to the shorter sequenceobtained in the first run. Definite assignments could not be made at 3of the amino acid residues in the sequence (residues 1, 6 and 11 fromthe N-terminal). A search of protein databases did not detect anysignificantly homologous sequences, suggesting that the purified factorwas a novel protein.

This initial N-terminal amino acid sequence data did not enable theisolation of cDNA clones using degenerate oligonucleotides as PCRprimers or probes for screening libraries. To facilitate theseapproaches, additional protein was purified in order to obtain internalamino acid sequence from proteolytic fragments. To obtain internal aminoacid sequence from neurturin, an additional 50 liters of CHO cellconditioned medium was purified using only the first 3 chromatographicsteps as outlined above, except that the gradient used to elute the Cu++Chelating Superose column was as follows: 0-60 mM glycine (4 ml), 60 mMglycine (10 ml), 60-300 mM glycine (32 ml). Fractions No. 20-23containing neurturin were concentrated to 25 μl by ultrafiltration(Amicon microcon 3, Amicon, Beverley, Mass.) and loaded on anon-reducing SDS polyacrylamide gel. After electrophoresis, the gel wasstained with Coomassie blue and the 25 kD neurturin band was excised.Neuturin was digested in the gel slice with endoproteinase Lys-C, andthe eluted proteolytic fragments were purified by reverse phase HPLC.Only one peak was observed upon HPLC separation of the eluted peptides,which yielded amino acid seuquence information for 23 cycles at the 1pmole signal level using the automated sequencer, (internal fragment P2,SEQ ID NO:5).

Amino acid analysis performed on 10% of the above sample beforesubjecting it to digestion had indicated that 150 pmoles of protein werepresent in the gel slice, consisting of 7.6% lysine and 19.5% arginine.The single low level peak from the Lys-C digestion suggested that thedigestion and elution of peptides were inefficient. The same gel slicewas redigested with trypsin and the eluted peptides separated by HPLC.Two peaks were observed on HPLC, resulting in the elucidation of twoadditional 10 residue amino acid sequences (4-5 pmole signal level,internal fragment P1, SEQ ID NO:4 and internal fragment P3, SEQ ID NO:6)that were distinct from the N-terminal and previous internal amino acidsequences. The in situ digestion, elution and purification of peptides,and peptide sequencing was performed by the W. M. Keck FoundationBiotechnology Resource Laboratory at Yale University according tostandard protocols for this service.

EXAMPLE 5

The following example illustrates the isolation and sequence analysis ofmouse and human neurturin cDNA clones.

Degenerate oligonucleotides corresponding to various stretches ofconfident amino acid sequence data were synthesized and used as primersin the polymerase chain reaction (PCR) to amplify cDNA sequences fromreverse transcribed mRNA. A forward primer (M1676; 5′-CCNACNGCNTAYGARGA,SEQ ID NO:50) corresponding to peptide sequence P2Xaa₁-Xaa₂-Val-Glu-Ala-Lys-Pro-Cys-Cys-Gly-Pro-Thr-Ala-Tyr-Glu-Asp-Xaa₃-Val-Ser-Phe-Leu-Ser-Valwhere Xaa₁ and Xaa₂ were unknown, Xaa₃ was Gln or Glu (SEQ ID NO:5) incombination with a reverse primer (M1677; 5′-ARYTCYTGNARNGTRTGRTA (SEQID NO:52) corresponding to peptide sequence P3(Tyr-His-Thr-Leu-Gln-Glu-Leu-Ser-Ala-Arg) (SEQ ID NO:6) were used toamplify a 69 nucleotide product from cDNA template derived from E21 ratand adult mouse brain. The PCR parameters were: 94° C. for 30 sec; 55°C. for 30 sec; 72° C. for 1 min for 35 cycles. The product was subclonedinto the Bluescript KS plasmid and sequenced. All nucleotide sequencingwas performed using fluorescent dye terminator technology permanufacturer's instructions on an Applied Biosystems automated sequencerModel #373 (Applied Biosystems, Foster City, Calif.). Plasmid DNA forsequencing was prepared using the Wizard Miniprep kit (Promega Corp.,Madison, Wis.) according to the manufacturer's instructions. Thesequence of the amplified product correctly predicted amino acidsequence data internal to the PCR primers.

Primers corresponding to the amplified sequence were used in combinationwith the degenerate primers in the rapid amplification of cDNA ends(RACE) technique (Frohman, M. A. Methods in Enzymology 218:340-356,1993) using the Marathon RACE kit (CLONTECH, Palo Alto, Calif.) per themanufacturer's instructions, except that first strand cDNA synthesis wascarried out at 50° C. using Superscript II reverse transcriptase(Gibco-BRL). Briefly, a double stranded adaptor oligonucleotide wasligated to the ends of double stranded cDNA synthesized from postnatalday 1 rat brain mRNA. Using nested forward neurturin PCR primers (M1676:5′-CCNACNGCNTAYGARGA, SEQ ID NO:50 and 1678;5′-GACGAGGGTCCTTCCTGGACGTACACA, SEQ ID NO:53) in combination withprimers to the ligated adapter supplied in the kit (AP1, AP2), the 3′end of the neurturin cDNA was amplified by two successive PCR reactions(1st: M1676 and AP1, using 94° C. for 30 sec, 55° C. for 30 sec and 72°C. for 2 min for 35 cycles; 2nd: M1678 and AP2 using 94° C. for 30 secand 68° C. for 2 min for 35 cycles). A 5′ portion of the rat neurturincDNA was obtained by two successive PCR reactions using the linkeredcDNA as template. The 1st reaction utilized primers M1677 (SEQ ID NO:52)and AP1; using 94° C. for 30 sec; 55° C. for 30 sec; and 72° C. for 2min for 35 cycles. The 2nd reaction used M16795′-TAGCGGCTGTGTACGTCCAGGAAGGACACCTCGT (SEQ ID NO:54) and AP2 at 94° C.for 30 sec and 68° C. for 2 min for 35 cycles. These reactions resultedin a truncated form of the 5′ end of the neurturin cDNA, apparently theresult of premature termination of the cDNA during reversetranscription. The 5′ and 3′ RACE products were subcloned into theplasmid Bluescript KS and sequenced. The sequence of these 3′ and 5′RACE products resulted in a partial rat neurturin cDNA sequence of 220nt. Primers (#467921 5′-CAGCGACGACGCGTGCGCAAAGAGCG, SEQ ID NO:55; andM1679 (SEQ ID NO:54) corresponding to the partial rat cDNA sequence wereused (PCR parameters 94° C. for 30 sec and 68° C. for 1 min for 35cycles) to amplify a 101 nucleotide PCR product from mouse genomic DNAwhich was homologous to rat neurturin cDNA sequence.

These primers were then used to obtain murine neurturin genomic clonesby amplifying gene fragments in a mouse 129/Sv library in a P1bacteriophage vector (library screening service of Genome Systems, Inc.,St. Louis, Mo.). A 1.6 kb Nco I fragment from this P1 clone containingthe neurturin gene was identified by hybridization with primer (#465782;5′-TAYGARGACGAGGTGTCCTTCCTGGACGTACACAGCCGCTAYCAYAC, SEQ ID NO:56).ThisNco I fragment was sequenced and found to contain a stretch of codingsequence corresponding to the N-terminal and internal amino acidsequences obtained from sequencing the active protein isolated from CHOcell conditioned media. Beginning at the N-terminal amino acid sequenceof the purified protein, this nucleotide sequence encodes a 100 aminoacid protein with a predicted molecular mass of 11.5 kD. A search ofprotein and nucleic acid databases identified neurturin as a novelprotein that is approximately 40% identical to glial derivedneurotrophic factor (GDNF). GDNF was purified and cloned as a factorwhich promotes the survival of midbrain dopaminergic neurons and is adistantly related member of the TGF-β superfamily, which now includesmore than 25 different genes that possess a wide variety ofproliferative and differentiative activities. Although GDNF is less than20% identical to any other member of the TGF-β family, it contains the 7cysteine residues which are conserved across the entire family andbelieved to be the basis of a conserved cysteine knot structure observedin the crystal structure determination of TGF-β2. Neurturin alsocontains these 7 cysteine residues, but like GDNF is less than 20%homologous to any other member of the TGF-β family. Thus, neurturin andGDNF appear to represent a subfamily of growth factors which havesignificantly diverged from the rest of the TGF-β superfamily.

To determine the sequence of the full length mouse neurturin cDNA, 5′and 3′ RACE PCR was performed as above for the rat, using nested primerspredicted from the mouse genomic sequence and cDNA from neonatal mousebrain. The 1st reaction for the 3′ end used primers: M17775′-GCGGCCATCCGCATCTACGACCGGG (SEQ ID NO:57) and AP1 at 94° C. for 30sec; 65° C. for 15 sec; and 68° C. for 2 min for 35 cycles. The 2ndreaction used primer #467921 (SEQ ID NO:55) and AP2 at 94° C. for 30sec; 65° C. for 15 sec; and 68° C. for 2 min for 20 cycles. The 5′ endwas obtained using for the 1st reaction primer M1759,5′-CRTAGGCCGTCGGGCGRCARCACGGGT (SEQ ID NO:58) and AP1 at 94° C. for 30sec; 65° C. for 15 sec; and 68° C. for 2 min for 35 cycles. The 2ndreaction used primer M1785, 5′-GCGCCGAAGGCCCAGGTCGTAGATGCG (SEQ IDNO:59) and AP2 at 94° C. for 30 sec; 65° C. for 15 sec; and 68° C. for 2min for 20 cycles. Both sets of PCR reactions included 5% DMSO. The 5′and 3′ mouse RACE products were subcloned into the plasmid Bluescript KSand sequenced. Using the sequence of RACE products, a 1.0 kb mouseneurturin cDNA sequence can be assembled. This cDNA sequence contains anopen reading frame of 585 nucleotides that encodes a protein with amolecular mass of 24 kD. This full length mouse cDNA sequence is shownin FIG. 7 (SEQ ID NO:12). Consistent with the processing events known tooccur for TGF-β family members, the 24 kD neurturin protein contains anamino terminal 19 amino acid signal sequence followed by a pro-domainwhich contains an RXXR proteolytic processing site immediately beforethe N-terminal amino acid sequence obtained when sequencing the proteinpurified from CHO cell conditioned media. Using these landmarks, the11.5 kD mature neurturin molecule is predicted to be 11.5 kD and, byanalogy to other members of the TGF-β family, is predicted to form adisulfide linked homodimer of 23 kD, consistent with the 25 kD mass ofthe protein purified from CHO cell conditioned media as estimated bySDS-PAGE analysis.

For isolation of human genomic clones, primers (#467524:5′-CGCTACTGCGCAGGCGCGTGCGARGCGGC, SEQ ID NO:60 and #10005,5′-CGCCGACAGCTCTTGCAGCGTRTGGTA, SEQ ID NO:61) predicted from thesequence of mouse neurturin were used to amplify (PCR parameters:Initial denaturation at 95° C. for 1 min 30 sec followed by 94° C. for30 sec; 60° C. for 15 sec; and 68° C. for 60 sec for 35 cycles) a 192nucleotide fragment from human genomic DNA. The sequence of the PCRproduct demonstrated that it was the human homolog of mouse neurturin.The primers were then used to screen a human genomic library constructedin the P1 vector (library screening service, Genome Systems, Inc.) andtwo clones containing the human neurturin genomic locus were obtained.

The same strategy was used to determine the human sequence as discussedabove for the mouse sequence. An oligo (#30152,GACCTGGGCCTGGGCTACGCGTCCGACGAG, SEQ ID NO:62) was used as a probe in aSouthern blot analysis to identify restriction fragments of the P1Clones which contained the human neurturin coding sequence. Theserestriction fragments (Eag I, Pvu II, Hind III, Kpn I) were subclonedinto the Bluescript KS plasmid and sequenced.

The results of subcloning and sequencing of human genomic fragments wereas follows. The Eag I fragment was found to be approximately 6 kb insize with the 3′ Eag I site located 60 bp downstream from the stopcodon. The Pvu III fragment was approximately 3.5 kb in size with the 3′Pvu II site located 250 bp downstream from the stop codon. The Hind IIIfragment was approximately 4.8 kb in size with the 3′ Hind III sitelocated 3 kb downstream from the stop codon. The Kpn I fragment wasapproximately 4.2 kb in size with the 3′ Kpn I site located 3.1 kbdownstream from the stop codon.

The second coding exon was sequenced using these subcloned fragments. Inaddition, sequence was obtained from 250 bp flanking the 3′ side of thesecond exon. The sequence was also obtained from 1000 bp flanking the 5′side of the coding exon. From these flanking sequences, forward primer30341 (5′-CTGGCGTCCCAMCAAGGGTCTTCG-3′, SEQ ID NO:71) and reverse primer30331 (5′-GCCAGTGGTGCCGTCGAGGCGGG-3′, SEQ ID NO:72) were designed sothat the entire coding sequence of the second exon could be amplified byPCR.

The first coding exon was not mapped relative to the restriction sitesabove but was contained in the Eag I fragment. The sequence of this exonwas obtained from the subcloned Eag I fragment using the mouse primer466215 (5′-GGCCCAGGATGAGGCGCTGGAAGG-3′, SEQ ID NO:73), which containsthe ATG initiation codon. Further sequence of the first coding exon wasobtained with reverse primer 20215 (5′-CCACTCCACTGCCTGAWATTCWACCCC-3′,SEQ ID NO:74), designed from the sequence obtained with primer 466215.Forward primer 20205 (5′-CCATGTGATTATCGACCATTCGGC-3′, SEQ ID NO:75) wasdesigned from sequence obtained with primer 20215. Primers 20205 and20215 flank the coding sequence of the first coding exon and can be usedto amplify this coding sequence using PCR.

EXAMPLE 6

This example illustrates the preparation of expression vectorscontaining neurturin cDNA.

For expression of recombinant neurturin in mammalian cells the neurturinvector pCMV-NTN-3-1 was constructed. The 585 nucleotide open readingframe of the neurturin cDNA was amplified by PCR using a primercontaining the first 27 nucleotides of the neurturin coding sequence(5′-GCGACGCGTACCATGAGCGCTGGAAGGCAGCGGCCCTG, SEQ ID NO:63) and a primercontaining the last 5 codons and the stop codon(5′-GACGGATCCGCATCACACGCACGCGCACTC) (SEQ ID NO:64) using reversetranscribed postnatal day 1 mouse brain mRNA as template using (PCRparameters: 94° C. for 30 sec; 60° C. for 15 sec; and 68° C. for 2 minfor 35 cycles and including 5% DMSO in the reaction). The PCR productwas subcloned into the Eco RV site of BSKS and sequenced to verify thatit contained no PCR generated mutations. The neurturin coding sequencewas then excised from this vector using Mlu I (5′ end) and Bam Hl (3′end) and inserted downstream of the CMV IE promotor/enhancer in themammalian expression vector pCB6 (Brewer, C. B. Methods in Cell Biology43:233-245, 1994) to produce the pCMV-NTN-3-1 vector using these sites.

For expression of recombinant protein in E. Coli, the mature codingregion of mouse neurturin was amplified by PCR using a primer containingthe first 7 codons of the mature coding sequence(5′-GACCATATGCCGGGGGCTCGGCCTTGTGG) (SEQ ID NO:65) and a primercontaining the last 5 codons and the stop codon5′-GACGGATCCGCATCACACGCACGCGCACTC (SEQ ID NO:66) using a fragmentcontaining the murine neurturin gene as template using (PCR parameters:94° C. for 30 sec; 6° C. for 15 sec and 68° C. for 90 sec for 25 cycleswith 5%=DMSO added into the reaction). The amplified product wassubcloned into the Eco RV site of BSKS, the nucleotide sequence wasverified, and this fragment was then transferred to the expressionvector pET-30a (Novagen, Madison, Wis.) using an Nde 1 site (5′ end) andan Eco Rl site (3′ end). The pET-neurturin (pET-NTN) vector codes for aninitiator methionine in front of the first amino acid of the maturemouse neurturin protein predicted from the N-terminal amino acidsequence of neurturin purified from the CHO cell conditioned media.

EXAMPLE 7

The example illustrates the transient transfection of NIH3T3 cells withthe neurturin expression vector pCMV-NTN-3-1 and that the product of thegenomic sequence in Example 5 is bilogically active.

To demonstrate that the cloned neurturin cDNA was sufficient to directthe synthesis of biologically active neurturin we transiently introducedthe pCMV-NTN-3-1 plasmid into NIH3T3 cells using the lipofectaminemethod of transfection. NIH3T3 cells were plated at a density of 400,000cells per well (34.6 mm diameter) in 6 well plates (Corning, Corning,N.Y.) 24 hours before transfection. DNA liposome complexes were preparedand added to the cells according to the manufacturer's protocol using1.5 μg CMV-neurturin plasmid DNA (isolated and purified using a Qiagen(Chatsworth, Calif.) tip-500 column according to manufacturer'sprotocol) and 10 μl lipofectamine reagent (Gibco BRL; Gaithersburg, Md.)in 1:1 DME/F12 meidum containing 5 μg/ml insulin, 5 μg/ml transferrin,and 5 ng/ml sodium selenite (Sigma, St. Louis, Mo.). Five hours afterthe addition of DNA liposome complexes in 1 ml medium per well, 1 ml DMEmedium containing 20% calf serum was added to each well. Twenty-fourhours after the addition of DNA-liposome complexes, the 2 ml mediumabove was replaced with 1 ml DME medium containing 10% calf serum, 2 mMglutamine, 100 U/ml penicillin, 100 μ/ml streptomycin, and 25 ug/mlheparin. The cells were incubated for an additional 24 hours before theconditioned medium was harvested, centrifuged to remove cellular debris,and frozen.

As a control, NIH3T3 cells were transfected as above using 1.5 μgCMV-neo expression plasmid (containing no cDNA insert) in place of the1.5 μg CMV-neurturin plasmid. Conditioned medium from NIH3T3 cellstransfected with either control plasmid or CMV-neurturin plasmid wasassayed by direct addition to the SCG culture medium at the time of NGFdeprivation. Addition of 0.25 ml conditioned medium fromCMV-neurturin-transfected cells promoted 70% survival of sympatheticneurons, and >90% survival could be obtained with 0.45 ml of thisconditioned medium. No significant survival promoting activity wasdetected in the conditioned medium of control transfected NIH3T3 cells.

EXAMPLE 8

This example illustrates the preparation of Chinese hamster ovary cellsstably transformed with neurturin cDNA.

DG44 cells, a Chinese hamster ovary cell derivative that is deficient indihydrofolate reductase (DHFR) (Urlaub et all Cell 3:405-412, 1993 whichis incorporated by reference), were stably co-transfected withexpression plasmid (pCMV-NTN-3-1) and a DHFR expression plasmid (HLD)(McArthur, and Stanners J. Biol. Chem. 266:6000-6005, 1991 which isincorporated by reference).

On day 1 DG44 cells were placed at 1×10⁶ cells per 10 cm plate in Ham'sF12 medium with 10% fetal calf serum (FCS). This density must not beexceeded or cells will overgrow before selection media is added on day5.

On day 2 cells were transfected with a 9:1 ratio of pCMV-NTN to DHFRexpression plasmid using the calcium phosphate method (10 ug DNA/10 cmplate) (Chen and Okayama, Mol Cell Biol 7:2745-2752, 1987 which isincorporated by reference).

On day 3 the transfected cells were washed with Ham's F12 medium and fedHam's F12 with 10% FCS.

On day 5 the cells were washed with MEM alpha medium and fed selectionmedium, which is MEM alpha with 10% FCS and 400 ug/ml G418. The cellswere maintained in selection media, feeding every 4 days. Colonies beganto appear approximately 14 days after transfection.

Colonies growing in selection media were then transferred to a 24 wellplate and trypsinized the next day to disperse the cells. The cells weregrown to confluence in either 24 well or 6 well plates in order toscreen the cells for expression of recombinant protein. Expression ofneurturin was examined in 10 clonal lines and two high expressing lineswere detected using the SCG survival assay. These clonal lines wereexpanded and expression in these selected cell lines was amplified byselection in 50 nM methotrexate (MTX). For selection in MTX, cells weregrown to 50% confluence in a 150 cm² flask in selection medium. Themedium was changed to MEM alpha containing 50 nM MTX concentration (itwas not necessary to use G418 during MTX amplification). After placementin 50 nM MTX, the majority of cells died and colonies of resistant cellsreappearEd in 1-2 weeks. At this time, the cells were trypsinized todisperse colonies and are split when cells reach confluence. Cellseventually reached the same growth rate as before. The selected cellswere screened for expresssion of recombinant protein. A 2-3 foldincrease in expression was observed after selection in 50 nM MTX. Frozenstocks were kept for cell lines obtained from the original selection andthe 50 nM MTX selection. Further selection could be continued inincreasing MTX until desired levels of expression are obtained.

Using the above method, we isolated cells identified as DG44CHO5-3(G418) (pCMV-NTN-3-1) and DG44CHO5-3 (50 nMMTX) (pCMV-NTN-3-1). Cellsfrom the DG44CHO5-3 (50 nMMTX) (pCMV-NTN-3-1) strain expressed levels ofapproximately 100 μg of biologically active protein per liter ofconditioned media determined by direct assay of conditioned medium inSCG assay according to the methods in example 1.

EXAMPLE 9

This example illustrates the expression of neurturin in various tissues.

A survey of neurturin and GDNF expression was performed in rat embryonictissues (E10, day 10 after conception), neonatal tissues (P1, PostnatalDay 1), and adult tissues (>3 mos) using semi-quantitative RT/PCR (Estuset al., J Cell Biol 127:1717-1727, 1994 which is incorporated byreference). The RNA samples were obtained from various tissues and PCRproducts were detected either by autoradiography after incorporation ofα-³²P-dCTP in the PCR and electrophoresis on a polyacrylamide gel (FIG.6) or by ethidium bromide staining of DNA after electrophoresis onagarose gels (Tables 3 and 4). The neurturin fragment of 101 base pairswas obtained using the forward primer CAGCGACGACGCGTGCGCAAAGAGCG (SEQ IDNO:67) and reverse primer TAGCGGCTGTGTACGTCCAGGAAGGACACCTCGT (SEQ IDNO:68) and the GDNF fragment of 194 base pairs was obtained using theforward primer AAAAATCGGGGGTGYGTCTTA (SEQ ID NO:69) and the reverseprimer CATGCCTGGCCTACYTTGTCA (SEQ ID NO:70).

No neurturin or GDNF mRNA was detected at the earliest embryonic age(embryonic day 10, E10) surveyed.

In neonates (postnatal day 1, P1) both transcripts were expressed inmany tissues although neurturin tended to show a greater expression inmost tissues than did GDNF. (see Table 3).

TABLE 3 NEURTURIN GDNF Liver +++ − Blood +++ + Thymus + − Brain ++ +Sciatic nerve − + Kidney ++ ++ Spleen ++ + Cerebellum ++ + Heart ++ +Bone + +

As shown in Table 3, difference in the tissue distributions of neurturinand GDNF were noted. In particular, no GDNF was detected in liver andthymus where neurturin expression was detected and no neurturin wasdetected in sciatic nerve where GDNF was detected.

Neurturin and GDNF mRNA were detected in many tissues in the adultanimal, but the tissue-specific pattern of expression for these twogenes was very different (table 4, FIG. 5).

TABLE 3 NEURTURIN GDNF Liver − − Blood + − Thymus + ++ Brain + − Sciaticnerve − − Kidney ++ + Spleen − + Cerebellum − − Uterus ++ − Bone marrow++ − Testis ++ ++ Ovary + + Placenta + − Skeletal + − muscle Spinalcord + − Adrenal ++ ++ gland Gut + ++

As shown in table 4, neurturin was found to be expressed in brain andspinal cord as well as in blood and bone marrow where no GDNF wasdetected. The level of expression of neurturin in brain and blood was,however, less than that detected in brain and blood was, however, lessthan that detected in neonatal tissue.

Neurturin was also highly expressed in freshly isolated rat peritonealmast cells, whereas GDNF showed little or no expression.

EXAMPLE 10

This example illustrates the preparation of antisera to neurturin byimmunization of rabbits with a neurturin peptide.

The peptide sequence corresponding to amino acids 73-87 of the maturemurine neurturin protein was synthesized and coupled to keyhole limpethemocyanin (KLH) as described earlier (Harlow and Lane, Antibodies: alaboratory manual, 1988. Cold Spring Harbor Laboratory, New York, N.Y.p. 72-81 which is incorporated by reference). The KLH-coupled peptidewas submitted to Caltag, Inc. and each of two rabbits were immunized.Immunization was by subcutaneous injection at 7-10 sites. The firstinjection was with 150 μg KLH-coupled peptide which was resuspended in0.5 ml saline and emulsified with 0.5 ml complete Freund's adjuvant.Boost injections were begun 4 weeks after the initial injection and wereperformed once every 7 days as above for a total of 5 injections exceptthat 100 μg of KLH-coupled peptide and incomplete Freund's adjuvant wereused. Serum samples were collected 1 week after the fifth boost.

A pooled volume of twenty ml of serum that had been collected from bothrabbits one week after the 5th injection was purified. For purification,a peptide affinity column was prepared by coupling the above peptide tocyanogen bromide activated Sepharose 4B according to the manufactureprotocol (Pharmacia Biotech). The serum was diluted 10 fold in 10 mMTris pH 7.5 buffer and mixed by gentle rocking for 16 hours at 4° C.with 0.5 ml of peptide agarose matrix containing 5 mg of coupledpeptide. The matrix was placed into a column, washed with 5 ml of 10 mMTris pH 7.5, 150 mM NaCl, washed with 5 ml of 10 mM Tris pH 7.5 buffercontaining 0.4 M NaCl and eluted with 5.5 ml of 100 mM glycine pH 2.5buffer. One tenth volume of 1.0M Tris pH 8.0 buffer was added to theeluate immediately after elution to neutralize the pH. The glycineeluate was dialyzed overnight against 10 mM Tris pH 7.5, 150 mM NaCl.

The affinity-purified antibodies were used in a western blot todemonstrate specific recognition of recombinant neurturin protein. Tenml of conditioned medium collected from DG44CHO5-3(G418) (pCMV-NTN-3-1)cells was purified over SP Sepharose as described in Example 1 and theproteins electrophoresed on a reducing SDS-PAGE gel in the tricinebuffer system (Schagger and von Jagow Analytical Biochemistry166:368-379, 1987). The proteins were electroblotted to a nitrocellulosemembrane in 25 mM Tris, 192 mM glycine, 0.04% SDS, 17% methanol at 4° C.for 16 hr. The membrane was incubated with the affinity-purifiedanti-neurturin peptide antibodies and then with horseradishperoxidase-coupled sheep anti-rabbit IgG (Harlow and Lane, supra, p.498-510). Bound antibodies were detected with enhanced chemiluminescence(ECL kit, Amersham, Buckinghamshire, England). The anti-neurturinantibodies recognized a single, approximately 11.5 kD protein band inthe conditioned medium of the DG44CHO5-3(G418) (pCMV-NTN-3-1) cells.Using these anti-neurturin antibodies, neurturin protein could bedetected in 10 ml of coniditioned medium from DG44CHO5-3 (G418)(pCMV-NTN-3-1) cells but could not be detected in 10 ml of mediumconditioned with DG44 cells that had not been transformed with theneurturin expression vector.

EXAMPLE 11

The following example illustrates the identification of additionalmembers of the GDNF/neurturin/persephin gene subfamily.

The TGF-β superfamily currently contains over 25 different gene members(for review see Kingsley, Genes and Development 8: 133-146, 1994 whichis incorporated by reference). The individual family members displayvarying degrees of homology with each other and several subgroups withinthe superfamily can be defined by phylogenetic analysis using theClustal V program (Higgins et al, Comput Appl Biosci 8: 189-191, 1992which is incorporated by reference) and by bootstrap analysis ofphylogenetic trees (Felsenstein, Evolution 39: 783-791, 1985 which isincorporated by reference). Neurturin or persephin is approximately 40%identical to GDNF but less than 20% identical to any other member of theTGF-β superfamily. Several sequence regions in neurturin can beidentified (FIG. 5) that are highly conserved within theGDNF/neurturin/persephin subfamily but not within the TGF-β superfamily.These conserved regions are likely to characterize a subfamilycontaining previously unisolated genes, which can now be isolated usingthe conserved sequence regions identified by the discovery andsequencing of the neurturin and persephin genes. Regions of highsequence conservation between neurturin, persephin and GDNF allow thedesign of degenerate oligonucleotides which can be used either as probesor primers. Conserved-region amino acid sequences have been identifiedherein to include Val-Xaa₁-Xaa₂-Leu-Gly-Leu-Gly-Tyr where Xaa₁ is Ser,Thr or Ala and Xaa₂ is Glu or Asp (SEQ ID NO:108);Glu-Xaa₁-Xaa₂-Xaa₃-Phe-Arg-Tyr-Cys-Xaa₄-Gly-Xaa₅-Cys in which Xaa₁ isThr, Glu or lys, Xaa₂ is Val, Leu or Ile, Xaa₃ is Leu or Ile, Xaa₄ isAla or Ser, and Xaa₅ is Ala or Ser, (SEQ ID NO:113); andCys-Cys-Xaa₁-Pro-Xaa₂-Xaa₃-Xaa₄-Xaa₅-Asp-Xaa₆-Xaa₇-Xaa₈-Phe-Leu-Asp-Xaa₉in which Xaa₁ is Arg or Gln, Xaa₂ is Thr or Val or Ile, Xaa₃ is Ala orSer, Xaa₄ is Tyr or Phe, Xaa₅ is Glu, Asp or Ala, Xaa₆ is Glu, Asp or noamino acid, Xaa₇ is val or leu, Xaa₈ is Ser or Thr, and Xaa₉ is Asp orVal (SEQ ID NO:114). Nucleotide sequences containing a coding sequencefor the above conserved sequences or fragments of the above conservedsequences can be used as probes. Exemplary probe and primer sequenceswhich can be designed from these regions are as follows.

Forward primers,

Primer A (M3119): 5′-GTNDGNGANYTGGGNYTGGGNTA (SEQ ID NO:115) 23 nt whichcodes for the amino acid sequence, Val-Xaa₁-Xaa₂-Leu-Gly-Leu-Gly-Tyrwhere Xaa₁ is Thr, Ser or Ala and Xaa₂ is Glu or Asp (SEQ ID NO:125);

Primer B (M3123): 5′-GANBTNWCNTTYYTNGANG (SEQ ID NO:116) 19 nt whichcodes for the amino acid sequence, Xaa₁-Xaa₂-Xaa₃-Phe-Leu-Xaa₄-Xaa₅where Xaa₁ is Asp or Glu, Xaa₂ is Val or Leu, Xaa₃ is Thr or Ser, Xaa₄is Asp or Glu, and Xaa₅ is Asp or Val (SEQ ID NO:126);

Primer C (M3126): 5′-GANBTNWCNTTYYTNGANGW (SEQ ID NO:117) 20 nt whichcodes for the amino acid sequence, Xaa₁-Xaa₂-Xaa₃-Phe-Leu-Xaa₄-Xaa₅where Xaa₁ is Asp or Glu, Xaa₂ is Val or Leu, Xaa₃ is Thr or Ser, Xaa₄is Asp or Glu, and Xaa₅ is Asp or Val (SEQ ID NO:126);

Primer D (M3121): 5′-TTYMGNTAYTGYDSNGGNDSNTG (SEQ ID NO:118) 23 nt whichcodes for the amino acid sequence, Phe-Arg-Tyr-Cys-Xaa₁-Gly-Xaa₂-Cyswhere Xaa₁ is Ser or Ala and Xaa₂ is Ser or Ala (SEQ ID NO:127);

Primer E (M3122): 5′-GTNDGNGANYTGGGNYTNGG (SEQ ID NO:119) 20 nt whichcodes for the amino acid sequence, Val-Xaa₁-Xaa₂-Leu-Gly-Leu-Gly whereXaa₁ is Thr, Ser or Ala and Xaa₂ is Asp or Glu (SEQ ID NO:128); and

Primer F (M3176): 5′-GTNDGNGANYTGGGNYTGGNTT (SEQ ID NO:120) 23 nt whichcodes for the amino acid sequence, Val-Xaa₁-Xaa₂-Leu-Gly-Leu-Gly-Phewhere Xaa₁ is Thr, Ser or Ala and Xaa₂ is Glu or Asp (SEQ ID NO:129).

Reverse primers,

Primer G (M3125): 5′-WCNTCNARRAANGWNAVNTC (SEQ ID NO:121) 20 nt whosereverse complementary sequence codes for the amino acid sequence,Xaa₁-Xaa₂-Xaa₃-Phe-Leu-Xaa₄-Xaa₅ where Xaa₁ is Asp or Glu, Xaa₂ is Valor Leu, Xaa₃ is Thr or Ser, Xaa₄ is Asp or Glu, and Xaa₅ is Asp or Val(SEQ ID NO:126);

Primer H (M3124): 5′-WCNTCNARRAANGWNAVNNT (SEQ ID NO:122) 19 nt whosereverse complementary sequence codes for the amino acid sequence,Xaa₁-Xaa₂-Xaa₃-Phe-Leu-Xaa₄-Xaa₅ where Xaa₁ is Asp or Glu, Xaa₂ is Valor Leu, Xaa₃ is Thr or Ser, Xaa₄ is Asp or Glu, and Xaa₅ is Asp or Val(SEQ ID NO:126);

Primer I (M3120): 5′-CANSHNCCNSHRCARTANCKRAA (SEQ ID NO:123) 23 NT whosereverse complementary sequence codes for the amino acid sequence,phe-Arg-Tyr-Cys-Xaa₁-Gly-Xaa₂-Cys where Xaa₁ is Asp or Glu, Xaa₂ is Valor Leu, Xaa₃ is Thr or Ser or Ala and Xaa₂ is Ser or Ala (SEQ IDNO:127); and

Primer J (M3118): 5′-CANSHNCCNSHRCARTANCKRAANA (SEQ ID NO:124) 25 NTwhose reverse complementary sequence codes for the amino acid sequence,Xaa₁-Phe-Arg-Tyr-Cys-Xaa₂-Gly-Xaa₃-Cys where Xaa₁ is Ile or Leu, Xaa₂ isSer or Ala and Xaa₃ is Ser or Ala (SEQ ID NO:130).

In addition to the above, the following primers are based upon conservedregions in GDNF and neurturin (SEQ ID NOS:33-35).

Primer 1, GNTWSNGANYTNGGNYTNGGNTA (SEQ ID NO:42) which encodes the aminoacid sequence, Val-Xaa₁-Xaa₂-Leu-Gly-Leu-Gly-Tyr where Xaa₁ is Ser orThr and Xaa₂ is Glu or Asp (SEQ ID NO:33);

Primer 2, TTYMGNTAYTGYDSNGGNDSNTGYGANKCNGC (SEQ ID NO:43) which encodesthe amino acid sequence Phe-Arg-Tyr-Cys-Xaa₁-Gly-Xaa₂-Cys-Xaa₃-Xaa₄-Alawhere Xaa₁ is Ala or Ser, Xaa₂ is Ala or Ser, Xaa₃ is Glu or Asp andXaa₄ is Ser or Ala (SEQ ID NO:36);

Primer 3 reverse GCNGMNTCRCANSHNCCNSHRTANCKRAA (SEQ ID NO:44) whosereverse complementary sequence encodes amino acid sequencePhe-Arg-Tyr-Cys-Xaa₁-Gly-Xaa₂-Cys-Xaa₃-Xaa₄-Ala where Xaa₁ is Ala orSer, Xaa₂ is Ala or Ser, Xaa₃ is Glu or Asp and Xaa₄ is Ser or Ala (SEQID NO:37);

Primer 4 reverse TCRTCNTCRWANGCNRYNGGNCKCARCA (SEQ ID NO:45) whosereverse complementary sequence encodes amino acid sequenceCys-Cys-Arg-Pro-Xaa₁-Ala-Xaa₂-Xaa₃-Asp-Xaa₄ where Xaa₁ is Ile or Thr orVal, Xaa₂ Try or Phe, Xaa₃ is Glu or Asp and Xaa₄ is Glu or Asp (SEQ IDNO:38);

Primer 5 reverse TCNARRAANSWNAVNTCRTCNTCRWANGC (SEQ ID NO:46) whosereverse complementary sequence encodes amino acid sequenceAla-Xaa₁-Xaa₂-Asp-Xaa₃-Xaa₄-Ser-Phe-Leu-Asp where where Xaa₁ is Tyr orPhe, Xaa₂ Glu or Asp, Xaa₃ is Glu or Asp, and Xaa₄ is Val or Leu (SEQ IDNO:39);

Primer 6 GARRMNBTNHTNTTYMGNTAYTG (SEQ ID NO:47) which encodes amino acidsequence Glu-Xaa₁-Xaa₂-Xaa₃-Phe-Arg-Tyr-Cys where Xaa₁ is Glu or Thr,Xaa₂ is Leu or Val and Xaa₃ is Ile or Leu (SEQ ID NO:40);

Primer 7 GARRMNBTNHTNTTYMGNTAYTGYDSNGGNDSNTGHGA (SEQ ID NO:48) whichencodes amino acid sequenceGlu-Xaa₁-Xaa₂-Xaa₃-Phe-Arg-Tyr-Cys-Xaa₄-Gly-Xaa₅-Cys-Xaa₆ where Xaa₁ isGlu or Thr, Xaa₂ is Leu or Val, Xaa₃ is Ile or Leu, Xaa₄ is Ser or Ala,Xaa₅ is Ser or Ala and Xaa₆ is Glu or Asp (SEQ ID NO:41).

The above sequences can be used as probes for screening libraries ofgenomic clones or as primers for amplifying gene fragments from genomicDNA or libraries of genomic clones or from reverse transcribed cDNAusing RNA templates from a variety of tissues. Genomic DNA or librariesof genomic clones can be used as templates because the neurturin,persephin and GDNF coding sequences for the mature proteins are notinterrupted by introns.

A degenerate oligonucleotide can be synthesized as a mixture ofoligonucleotides containing all of the possible nucleotide sequenceswhich code for the conserved amino acid sequence. To reduce the numberof different oligonucleotides in a degenerate mix, an inosine oruniversal base (Loakes et al, Nucleic Acids Res 22:4039-43, 1994) can beincorporated in the synthesis at positions where all four nucleotidesare possible. The inosine or universal base forms base pairs with eachof the four normal DNA bases which are less stabilizing than AT and GCbase pairs but which are also less destabilizing than mismatches betweenthe normal bases (i.e. AG, AC, TG, TC).

To isolate family members a primer above can be end labeled with ³²Pusing T4 polynucleotide kinase and hybridized to libraries of humangenomic clones according to standard procedures.

A preferred method for isolating family member genes would be to usevarious combinations of the degenerate primers above as primers in thepolymerase chain reaction using genomic DNA as a template. The variouscombinations of primers can include sequential PCR reactions utilizingnested primers or the use of a forward primer paired with an oligo dTprimer. In addition, one of the degenerate primers can be used with avector primer, a single primer can be used in an inverted PCR assay orPCR can be performed with one degenerate primer and a random primer. Asan example using the above set of primers, primer 2(SEQ ID NO:43) can beused with primer 4 (SEQ ID NO:45) in PCR with 1 ug of human genomic DNAand cycling parameters of 94° C. for 30 sec, 50° C. for 30 sec, and 72°C. for 60 sec. The above PCR conditions are exemplary only and oneskilled in the art will readily appreciate that a range of suitableconditions and primer combinations could be used or optimized such asdifferent temperatures and varying salt concentrations in the buffermedium and the like. It is preferred that DMSO be added to the PCRreaction to a final concentration of 5% inasmuch as this was found to benecessary for amplification of this region of the neurturin gene. ThePCR reaction, when run on an agarose gel, should contain products in thesize range of 100-150 base pairs since a one amino acid gap isintroduced in the neurturin sequence and a five amino acid gap isintroduced in the persephin sequence when either sequence is alignedwith GDNF, and thus family member genes might also contain a slightlyvariable spacing between the conserved sequences of primers 2 and 4. ThePCR products in the range of 100-150 base pairs should contain multipleamplified gene products including GDNF, neurturin and persephin as wellas previously unisolated family members. To identify sequences of theseproducts, they can be gel purified and ligated into the Bluescriptplasmid (Stratagene), and then transformed into the XL1-blue E. Colihost strain (Stratagene). Bacterial colonies containing individualsubclones can be picked for isolation and plated on nitrocellulosefilters in two replicas. Each of the replicate filters can be screenedwith an oligonucleotide probe for either unique GDNF or unique neurturinor unique persephin sequence in the amplified region. Subclones nothybridizing to either GDNF or neurturin or persephin can be sequencedand if found to encode previously unisolated family members, thesequence can be used to isolate full length cDNA clones and genomicclones as was done for neurturin (Example 5). A similar method was usedto isolate new gene members (GDF-3 and GDF-9) of the TGF-β superfamilybased on homology between previously identified genes (McPherron J BiolChem 268: 3444-3449, 1993 which is incorporated by reference).

The inventors herein believe that the most preferred way to isolatefamily member genes may be to apply the above PCR procedure as ascreening method to isolate individual family member genomic clones froma library. This is because there is only one exon for the coding regionof both mature neurturin and GDNF. If, for example, the above PCRreaction with primers 2 and 4 generates products of the appropriate sizeusing human genomic DNA as template, the same reaction can be performedusing, as template, pools of genomic clones in the P1 vector accordingto methods well known in the art, for example that used for isolatingneurturin human genomic clones (Example 5). Pools containing theneurturin gene in this library have previously been identified andpesephin and GDNF-containing pools can be readily identified byscreening with GDNF specific primers. Thus non-neurturin, non-persephin,non-GDNF pools which generate a product of the correct size using thedegenerate primers will be readily recognized as previously unisolatedfamily members. The PCR products generated from these pools can besequenced directly using the automated sequencer and genomic clones canbe isolated by further subdivision and screening of the pooled clones asa standard service offered by Genome Systems, Inc.

EXAMPLE 12

The following example illustrates the isolation and identification ofpersephin utilizing the procedures and primers described in Example 11.

The degenerate PCR strategy devised by the inventors herein has now beensuccessfully utilized to identify a third factor, persephin, that isapproximately 35-50% identical to both GDNF and neurturin. Theexperimental approach was described above and is provided in greaterdetail as follows. Primers corresponding to the amino acid sequenceVal-Xaa1-Xaa2-Leu-Gly-Leu-Gly-Tyr where Xaa1 is Ser or Thr and Xaa2 isGlu or Asp (SEQ ID NO:33) [M1996; 5′-GTNWSNGANYTNGGNYTNGGNTA (SEQ IDNO:42)] and Phe-Arg-Tyr-Cys-Xaa1-Gly-Xaa2-Cys-Xaa3-Xaa4-Ala where Xaa1is Ala or Ser, Xaa2 is Ala or Ser, Xaa3 is Glu or Asp and Xaa4 is Ser orAla (SEQ ID NO:37) [M1999; 5′-GCNGMNTCRCANSHNCCNSHRCARTANCKRAA (SEQ IDNO:44)] were used to amplify a 77 nt fragment from rat genomic DNA usingKlentaq enzyme and buffer under the following conditions: 94° C. for 30sec; 44° C. for 30 sec; 72° C. for 30 sec for 40 cycles. The resultingproduct was subcloned into the Bluescript KS plasmid and sequenced. Allnucleotide sequencing was performed using fluorescent dye terminatortechnology per manufacturer's instructions on an Applied Biosystemsautomated sequencer Model #373 (Applied Biosystems, Foster City,Calif.). Plasmid DNA for sequencing was prepared using the WizardMiniprep kit (Promega Corp., Madison, Wis.) according to themanufacturer's instructions.

The sequence of one of the amplified products predicted amino acidsequence data internal to the PCR primers that was different from thatof GDNF or neurturin but had more than 20% identity with GDNF andneurturin, whereas the sequences of others we obtained corresponded toGDNF or neurturin, as would be expected. The novel sequence was thoughtto identify a new member of this family which we named persephin.

The sequence of this fragment internal to the primers was5′-TGCCTCAGAGGAGAAGATTATC (SEQ ID NO:90). This encodes the lastnucleotide of the Tyr codon, and then encodes the amino acids:Ala-Ser-Glu-Glu-Lys-Ile-Ile (SEQ ID NO:91). This sequence was thenaligned with the rat sequences of GDNF and neurturin. This analysisconfirmed that persephin was unique.

LGLGYETKEELIFRYC GDNF (rat) (SEQ ID NO:92)

LGLGYTSDETVLFRYC NTN (rat) (SEQ ID NO:93)

LGLGYASEEKIIFRYC PSP (rat) (SEQ ID NO:94)

To obtain additional persephin sequence, primers containing portions ofthe unique 22 nt of the amplified fragment above were used in the rapidamplification of cDNA ends (RACE) technique (Frohman, M. A. Methods inEnzymology 218:340-356, 1993) using the Marathon RACE kit (CLONTECH,Palo Alto, Calif.) per the manufacturer's instructions, except thatfirst strand cDNA synthesis was carried out at 50° C. using SuperscriptII reverse transcriptase (Gibco-BRL). Briefly, a double stranded adaptoroligonucleotide was ligated to the ends of double stranded cDNAsynthesized from postnatal day 1 rat brain mRNA. Using nested forwardpersephin PCR primers, (10135; 5′-AGTCGGGGTTGGGGTATGCCTCA, SEQ ID NO:95and M2026; 5′-TATGCCTCAGAGGAGAAGATTATCTT SEQ ID NO:96) in combinationwith primers to the ligated adaptor supplied in the kit (AP1, AP2), the3′ end of the persephin cDNA was amplified by two successive PCRreactions (1st: 10135 and AP1, using 94° C. for 30 sec, 60° C. for 15sec and 68° C. for 2 min for 35 cycles; 2nd: M2026 and AP2 using 94° C.for 30 sec, 60 for 15 sec and 68° C. for 2 min for 21 cycles). Anapproximately 350 nt fragment was obtained from this PCR reaction andthis fragment was directly sequenced using primer M2026. The sequence ofthis 3′ RACE product resulted in a partial rat persephin cDNA sequenceof approximately 350 nt (SEQ ID NO:97). The predicted amino acidsequence of this cDNA was compared to that of GDNF and neurturin, andfound to be approximately 40% homologous to each of these proteins.Importantly, the characteristic spacing of the cyteine residues inmembers of the TGF-β superfamily was present. Furthermore, in additionto the region of similarity encoded by the degenerate primers used toisolate persephin, another region of high homology shared between GDNFand neurturin, but absent in other members of the TGF-β superfamily, wasalso present in persephin

GDNF ACCRPVAFDDDLSFLDD (aa 60-76) (SEQ ID NO:98)

NTN PCCRPTAYEDEVSFKDV (aa 61-77) (SEQ ID NO:99)

PSP PCCQPTSYAD-VTFLDD (aa 57-72) (SEQ ID NO:100)

(Amino acid numbering uses the first Cys residue as amino acid 1).

With the confirmation that persephin was indeed a new member of theGDNF/neurturin subfamily, we isolated murine genomic clones of persephinto obtain additional sequence information. Primers (forward, M2026;5′-TATGCCTCAGAGGAGAAGATTATCTT, SEQ ID NO:96 and reverse, M3028;5′-TCATCAAGGAAGGTCACATCAGCATA, SEQ ID NO:101) corresponding to rat cDNAsequence were used in a PCR reaction (PCR parameters: 94° C. for 30 sec,55° C. for 15 sec and 72° C. for 30 sec for 35 cycles) to amplify a 155nt fragment from mouse genomic DNA which was homologous to rat persephincDNA sequence. These primers were then used to obtain murine persephingenomic clones from a mouse 129/Sv library in a P1 bacteriophage vector(library screening service of Genome Systems, Inc., St. Louis, Mo.).

Restriction fragments (3.4 kg Nco I and a 3.3 kb Bam H1) from this P1clone containing the persephin gene were identified by hybridizationwith a 210 nt fragment obtained by PCR using mouse genomic DNA withprimers (forward, M2026; SEQ ID NO:96 and reverse, M3159;5′-CCACCACAGCCACAAGCTGCGGSTGAGAGCTG, SEQ ID NO:102) and PCR parameters:94° C. for 30 sec, 55° C. for 15 sec and 72° C. for 30 sec for 35cycles. The Nco I and Bam H1 fragments were sequenced and found toencode a stretch of amino acids corresponding to that present in the ratpersephin RACE product, as well as being homologous to the matureregions of both neurturin and GDNF (FIG. 11).

When the amino acid sequences of murine GDNF, neurturin and persephinare aligned using the first cysteine as the starting point (which isdone because alterations in the cleavage sites between family memberscreates variability in the segments upstream of the first cysteine),persephin (91 amino acids) is somewhat smaller than either neurturin (95amino acids) or GDNF (94 amino acids). The overall identity within thisregion is about 50% with neurturin and about 40% with GDNF (FIG. 12).

Further nucleotide sequencing of the murine persephin Nco I fragmentrevealed the nucleotide sequence of the entire murine persephin gene(SEQ ID NO:131, FIG. 17). An open reading frame extends up to a stopcodon at positions 244-246. However, somewhere in this sequence there isan apparent anomaly such that the sequence encoding the RXXR cleavagesite (nucleotides at positions 257-268) and the sequence correspondingto the mature persephin protein (positions 269-556) are not co-linearwith this open reading frame. Instead, a second reading frame encodesthe cleavage site and the mature persephin. The two cogent readingframes are shown in FIG. 17.

Additional sequencing of the rat persephin has also been performed. Ratgenomic fragments were amplified by PCR using Klentaq and rat genomicDNA as a template. The forward primer #40266(5′-AATCCCCAGGACAGGCAGGGAAT; SEQ ID NO:137) corresponding to a regionupstream of the mouse persephin gene and a reverse primer M3156(5′-CGGTACCCAGATCTTCAGCCACCACAGCCACAAGC, SEQ ID NO:138) corresponding toa region within the mature rat persephin sequence were used with thefollowing parameters (95° C. for 15 sec, 55° C. for 15 sec, 68° C. for45 sec×30 cycles). The amplified product was kinased with T4polynucleotide kinase, the ends were blunted with E. coli DNA polymeraseI (Klenow fragment), and cloned into BSKS plasmid.

Nucleotide sequencing was performed to establish the sequence of theentire rat persephin gene (SEQ ID NO:134; FIG. 18). An open readingframe was found to extend from the sequence coding for an initiatormethionine up to a stop codon at positions 244-246 as was seen withmurine persephin. As was also seen with murine persephin, an anomaly wasfound to occur between the sequence encoding the initiator methionineand that encoding the cleavage site for the mature rat persephin suchthat two cogent reading frames exist as indicated in FIG. 18.Irrespective of this anomaly, mammalian cells express persephin fromeither the murine or rat full length genomic sequence as illustratedbelow.

EXAMPLE 13

This example illustrates the preparation of a bacterial expressionvector for murine persephin and its introduction into an E. Coli forexpression of recombinant mature persephin.

The persephin polynucleotide encoding the mature murine persephinprotein which begins 5 amino acids upstream of the first framework Cysresidue (SEQ ID NO:80) was cloned into the pET expression vector pET-30aat the Nde I and Bgl II sites. This persephin polynucleotide wasgenerated by PCR using the murine persephin P1 genomic clone as atemplate. A forward primer M3157(5′-GGACTATCATATGGCCCACCACCACCACCACCACCACGACGACGACAAGGCCTTGGCTGGTTCATGCCGA, SEQ ID NO:139) encoding an Nde I site, 8 histidineresidues, and an enterokinase site, and a reverse primer M3156(5′-CGGTACCCAGATCTTCAGCCACCACAGCCACAAGC, SEQ ID NO:138), whichcorresponds to the sequence encoding the last 6 amino acid residues ofthe mature persephin sequence, the stop codon and a Bgl II site, wereused. The PCR reaction conditions were 95° C. for 15 sec, 55° C. for 15sec, 68° C. for 60 sec×25 cycles. This PCR product was subcloned intothe EcoRV site of BSKS plasmid and sequenced to verify that it containedno mutations. The persephin sequence was then excised from this vectorusing Nde I and Bgl II and cloned into the Nde I (5′) and Bgl II (3′)sites of the bacterial expression vector pET30a (Novagen, Madison,Wis.). This expression vector would, therefore, produce the mature formof the persephin protein possessing an amino terminal tag consisting of8 histidine residues followed directly by an enterokinase site.

The plasmid was introduced into E. coli strain BL21 (DE3). To producepersephin, bacteria harboring this plasmid were grown for 16 hr,harvested, and lysed using 6M guanidine-HCl, 0.1 M NaH₂PO₄, 0.01 M Trisat pH 8.0, and recombinant persephin protein was purified from theselysates via chromatography over a Ni-NTA resin (Qiagen). The protein waseluted using 3 column volumes of Buffer E containing 8 M urea, 0.1 MNaH₂PO₄, 0.01 M Tris, at pH 4.5. The persephin was then renatured bydialysis in renaturation buffer consisting of 0.1 M NaH₂PO₄, 0.01 M Trisat pH 8.3, 0.15 M NaCl, 3 mM cysteine, 0.02% Tween-20, 10% glycerol andcontaining decreasing concentrations of urea beginning with 4 M for 16hr, followed by 2 M for 16 hr, 1M for 72 hr, and 0.5 M for 16 hr. Thepersephin concentration was then determined using a Dot Metric assay(Geno Technology, St. Louis, Mo.) and stored at 4° C.

This bacterially produced recombinant persephin was used as an immunogenin rabbits to produce antibodies to mature persephin. All of theimmunogen injections and blood drawing were performed at Cal Tag Inc.Healdsburg, Calif.). The anti-persephin antiserum was demonstrated tospecifically recognize persephin, but not neurturin or GDNF, usingprotein blot analysis. This persephin-specific antiserum was then usedto detect persephin in lysates prepared from transfected COS cells.

EXAMPLE 14

This example illustrates the preparation of mammalian expression vectorscontaining the murine or rat persephin genes and their incorporationinto mammalian cell lines for the production of mature persephin. Toconstruct the murine plasmid, a P1 clone containing the murine persephingene was used as a template in a PCR assay. Primers were designed suchthat the resulting polynucleotide would contain the persephin geneextending from the initiator Methionine codon to the stop codon 3′ tothe mature persephin coding sequence (SEQ ID NO:131). The PCR reactionutilized a forward primer M3175 (5′-TGCTGTCACCATGGCTGCAGGAAGACTTCGGA,SEQ ID NO:140) and reverse primer M3156(5′-CGGTACCCAGATCTTCAGCCACCACAGCCACAAGC, SEQ ID NO:138). To constructthe analogous rat plasmid, rat genomic DNA was used as a template in aPCR assay. The PCR reaction utilized a forward primer M3175(5′-TGCTGTCACCATGGCTGCAGGAAGACTTCGGA, SEQ ID NO:138). Both PCR reactionswere carried out using Klentaq and the following parameters: 95° C. for15 sec, 55° C. for 15 sec, 68° C. for 45 sec×25 cycles. The amplifiedproducts were kinased with T4 polynucleotide kinase, the ends wereblunted with E. coli DNA polymerase I (Klenow fragment), and cloned intoBSKS plasmid. Nucleotide sequencing was performed to verify that thecorrect clone was obtained. The rat and murine persephin polynucleotideswere excised using Sma I and Hind III and each cloned into a Asp718(blunted) and Hind III sites of the mammalian expression vector pCB6.

COS monkey cells were transfected with either the rat or murinepersephin expression vectors (16 μg per 5×10⁵ cells) or thenon-recombinant vector (pCB6) itself using the calcium phosphateprecipitation method (Chen and Okayama, Mol Cell Biol 7:2745-2752, 1987which is incorporated by reference). Forty eight hr later the cells werelysed in IP buffer containing 50 mM Tris at pH 7.5, 300 mM NaCl, 1%Triton X-100, 1% deoxycholate, 10 mM EDTA, 0.1% SDS, 5 μg/ml leupeptin,7 μg/ml pepstatin, and 250 μM PMSF. The samples were loaded onto a 15%SDS-polyacrylamide gel and the proteins were separated byelectrophoresis. The proteins were then transferred to nitrocellulose byelectroblotting. This nitrocellulose membrane was incubated withanti-persephin antibodies to detect the presence of persephin in thelysates.

As is shown in FIG. 19, lysates from cells transfected with either therat or murine persephin expression vectors, but not the lysate fromcells transfected with pCB6, contain high amounts of persephin. the sizeof the persephin detected was approximately 14 kD which is consistentwith the size predicted for the processed, i.e. mature form ofpersephin. This demonstrates that both the murine and rat persephingenes are capable of directing the synthesis of a properly processedpersephin molecule.

EXAMPLE 15

The following example illustrates the methods that can be used forisolation and identification of human persephin.

The identification of murine and rat persephin sequences now allows usto identify and isolate the human persephin gene. Due to the highconservation between human and rodent GDNF (approximately 95% identity)and between human and rodent neurturin (approximately 90% identity), itis believed that a similarly close relationship (i.e. greater than 85%identity) will be present between rodent and human persephin.

Different strategies can be used to obtain the human persephin gene. Inone preferred strategy, human genomic and cDNA libraries are screened byhybridization to the murine and/or the rat persephin sequences that havebeen identified herein (SEQ ID NOS:79-83) or portions of thesesequences. These DNA sequences or probes are labeled as described above,for example, with 32P-dCTP using either random priming or polynucleotidekinase. Hybridization conditions are described above and variousstringency conditions of hybridization are used. Stringency ofhybridization is determined by a number of factors during hybridizationand during the washing procedure, including temperature, ionic strength,length of time and concentration of formamide. These factors areoutlined in, for example, Sambrook et al. supra.

In an alternate preferred strategy, primers corresponding to portions ofthe rat or the murine persephin nucleotide sequence (or derivativesthereof) are employed in a PCR reaction using either human genomic DNAor cDNA reverse transcribed from RNA isolated from human tissue. As anexample, forward primer M2026 (SEQ ID NO:96) and reverse primer, M3028(SEQ ID NO:101) can be used in a PCR reaction using various conditionsas described above and human DNA templates to amplify a human persephinfragment. Primers that amplify such a fragment as confirmed bynucleotide sequencing of the fragment are then used to obtain humanpersephin clones. The clones are identified by virtue of their producingthe same amplified fragment following PCR with the selected primers in ahuman genomic library in a P1 bacteriophage vector (library screeningservice of Genome Systems, Inc., St. Louis, Mo.).

After obtaining positive clones by either of the methods above, thesehuman persephin clones are isolated and fragments are subcloned intoBluescript KS plasmids and sequenced. Nucleotide sequencing is performedusing fluorescent dye terminator technology on an Applied Biosystemsautomated sequencer Model #373 (Applied Biosystems, Foster City, Calif.)according to manufacturer's instructions. Plasmid DNA for sequencing isprepared using the Wizard Miniprep kit (Promega Corp., Madison, Wis.)according to manufacturer's instructions. Sequences of these humanfragments which are orthologous to the rat and murine persephinsequences are then identified and the full length nucleotide sequence ofhuman persephin is established from the sequences of these fragments.

EXAMPLE 16

This example illustrates the preparation of chimeric or hybridpolypeptide molecules that contain portions derived from persephin (PSP)and portions derived from neurturin (NTN).

As closely related members of the TGFβ family, each of persephin andneurturin is predicted to have a very similar overall structure, yetwhile neurturin promotes the survival of sympathetic neurons, theclosely related persephin does not. Two chimeras were produced byessentially replacing portions of persephin with neurturin, with thecrossover point located between the two adjacent, highly conserved thirdand fourth cysteine residues. The first chimera, named PSP/NTN (SEQ IDNO:141, FIG. 20), contains the first 63 residues of mature murinepersephin combined with residues 68 through 100 of mature murineneurturin (using E. coli preferred codons). To construct this molecule,two PCR reactions were performed: 1) using the forward primer M2012(5′-TAATACGACTCACTATAGGGGAA, SEQ ID NO:142) and reverse primer M2188(5′-TCGTCTTCGTAAGCAGTCGGACGGCAGCAGGGTCGGCCATGGGCTCGAC, SEQ ID NO:143)and the pET30a-murine persephin plasmid as template (see Example 13);and 2) using the forward primer M2190 (5′-TGCTGCCGTCCGACTGCTTACGAAGACGA,SEQ ID NO:144) and reverse primer M2186 (5′-GTTATGCTAGTTATTGCTCAGCGGT,SEQ ID NO:145) and the pET30a-murine (E. coli preferred codons)neurturin plasmid as template (see Example 6). Both PCR reactions werecarried out using the following parameters: 94° C. for 30 sec, 55° C.for 30 sec, 72° C. for 30 sec×25 cycles. The products of these two PCRreactions were gel purified, mixed together, and a PCR reaction wasperformed under the following conditions: 94° C. for 30 sec, 60° C. for20 min, 68° C. for 5 min. After 8 cycles, an aliquot of this reactionwas used as template in a third PCR reaction using the forward primerM2012 and reverse primer M2186 under the following conditions: 94° C.for 30 sec, 55° C. for 30 sec, 72° C. for 30 sec×25 cycles. Theresulting product was kinased with T4 polynucleotide kinase, the endswere blunted with E. coli DNA polymerase I (Klenow fragment), and clonedinto BSKS plasmid. Nucleotide sequencing was performed to verify thatthe correct clone was obtained. The PSP/NTN fragment was excised usingNde I and Bam H1 and cloned into the corresponding sites of thebacterial expression vector pET30a.

The second chimera, named NTN/PSP (SEQ ID NO:146, FIG. 20), encodes theconverse molecule. It contains the first 67 residues of mature murineneurturin (using E. coli preferred codons) combined with residues 64 to96 of mature murine persephin. To construct this molecule, we performedtwo PCR reactions: 1) using the forward primer M2012 and reverse primerM2183 (5′-CACATCAGCATAGCTGGTGGGCTGGCAGCACGGGTGAGCACGAGCACGTT, SEQ IDNO:147) and the pET30a-murine (E. coli preferred codons) neurturinplasmid as template; and 2) using the forward primer M2187(5′-TGCTGCCAGCCCACCAGCTATGCTG, SEQ ID NO:148) and reverse primer M2186(5′-GTTATGCTAGTTATTGCTCAGCGGT, SEQ ID NO:145) and the pET30a-murinepersephin plasmid as template. Both PCR reactions were carried out usingthe following parameters: 94° C. for 30 sec, 55° C. for 30 sec, 72° C.for 30 sec×25 cycles. The products of these two PCR reactions were usedto construct the final NTN/PSP pET30a plasmid as detailed above forPSP/NTN except that Bgl II was used instead of Bam H1. These chimericproteins were produced in E. coli and purified by Ni-NTA chromatographyas described above (Example 13).

The purified proteins were assayed for their ability to promote survivalin the SCG sympathetic neuron assay. The NTN/PSP protein did not promotesurvival, whereas the PSP/NTN protein promoted the survival ofsympathetic neurons similar to that observed for neurturin itself. Theseresults indicate that neurturin residues lying downstream of the 2adjacent, highly conserved cysteine residues are critical for activityin promoting survival in SCG sympathetic neurons. In contrast, thecorresponding residues of persephin are not sufficient for promotingsurvival in sympathetic neurons.

EXAMPLE 17

This example illustrates the neuronal survival promoting activity ofpersephin in mesencephalic cells.

The profile of survival promoting activity of persephin is differentfrom that of neurturin and GDNF. In contrast to the survival promotingactivity produced by neurturin and GDNF in sympathetic and sensoryneurons, persephin showed no survival promoting activity in thesetissues. We further evaluated the neuronal survival promoting activityof persephin in mesencephalic cells.

Timed-pregnant Sprague-Dawley rats were purchased from HarlanSprague-Dawley. The mesencephalon was taken from rats measuring 1.2 to1.4 cm in length and time dated to be embryonic day 14. The cranium wasremoved and the entire mesencephalon was placed in cold L15. The pooledmesencephalic tissue was resuspended in a serum-free medium consistingof DME/Hams F12 (#11330-032, Life Technologies) 1 mg/ml BSA, Fraction V(A-6793, Sigma Chemical Co.,), 5 μM Insulin (I-5500, Sigma), 10 nMprogesterone (PO130, Sigma), 100 μM putrescine, (p7505, Sigma), 30 nMSelenium (SO7150, Pflatz & Bauer), 10 ng/ml rat transferrin(012-000-050, Jackson Chrompure), 100 U/ml penicillin, and 100 U/ml ofstreptomycin. The pooled mesencephalic tissues were trituratedapproximately 80 times using a bent-tip pipette and the cells wereplated in a 24-well dish (CoStar) at a density of 15,000 cells in a100-μl drop. The dishes were coated with 125 ng/ml poly-d-lysine(p-7280, Sigma) and 25 ng/ml laminin (#40232, Collaborative BiomedicalProducts). These dissociated cells were allowed to attach for 2 hours at37° C. in 5% CO₂ and then fed with another 500 μl of the aboveserum-free medium with or without approximately 100 ng/ml of recombinantPersephin. These cells were photographed after 3 days of culture.

Inspection of the cells over the course of 3 days in culture, showed agradual decrease in cell number. In the absence of any growth factor,almost all of the cells were dead (FIG. 21A). In the presence ofpersephin, a large increase in mesencephalic neuronal cell survival wasevident (FIG. 21B).

EXAMPLE 18

This example illustrates the expression of persephin in various tissues.

A survey of persephin expression was performed in adult mouse tissuesusing semi-quantitative RT/PCR (see Example 9). Poly A RNA was isolatedfrom brain, cerebellum, kidney, lung, heart, ovary, sciatic nerve,dorsal root ganglia, blood and spleen. This was then reverse transcribedto produce cDNA (see Kotzbauer et al. Nature 384:467-470, 1996 which isincorporated by reference). The PCR primers used were as follows:forward primer: 5′-CCTCGGAGGAGAAGGTCATCTTC (SEQ ID NO:149) and reverseprimer: 5′TCATCAAGGAAGGTCACATCAGCATA (SEQ ID NO:101). PCR was done for26 cycles with an annealing temperature of 60° C. To control for thepresence of genomic DNA, RNA samples which were not reverse transcribedwere used for PCR (for example, the tissue control shown in FIG. 22 islabeled “Kidney no RT”). All the samples were found to be withoutgenomic DNA contamination.

As shown in FIG. 22, a band of the correct size (160 bp) was seen in thekidney sample. At higher cycle numbers a persephin band was also seen inbrain. Thus, the distribution of expression of persephin in variousmouse tissues differs from that of neurturin in rat (Example 8).

Deposit of Strain. The following strain is on deposit under the terms ofthe Budapest Treaty, with the American Type Culture Collection, 12301Parklawn Drive, Rockville, Md. The accession number indicated wasassigned after successful viability testing, and the requisite fees werepaid. Access to said cultures will be available during pendency of thepatent application to one determined by the Commissioner to be entitledthereto under 37 CFR 1.14 and 35 USC 122. All restriction onavailability of said cultures to the public will be irrevocably removedupon the granting of a patent based upon the application. Moreover, thedesignated deposits will be maintained for a period of thirty (30) yearsfrom the date of deposit, or for five (5) years after the last requestfor the deposit, or for the enforceable life of the U.S. patent,whichever is longer. Should a culture become nonviable or beinadvertently destroyed, or, in the case of plasmid-containing strains,lose its plasmid, it will be replaced with a viable culture. Thedeposited materials mentioned herein are intended for convenience only,and are not required to practice the present invention in view of thedescription herein, and in addition, these materials are incorporatedherein by reference.

Strain Deposit Date ATCC No. DG44CHO-pHSP-NGFI-B Aug. 25, 1995 CRL 11977

In view of the above, it will be seen that the several advantages of theinvention are achieved and other advantageous results attained.

As various changes could be made in the above methods and compositionswithout departing from the scope of the invention, it is intended thatall matter contained in the above description and shown in theaccompanying drawings shall be interpreted as illustrative and not in alimiting sense.

SEQUENCE LISTING (1) GENERAL INFORMATION: (iii) NUMBER OF SEQUENCES: 176(2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 102 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: unknown(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 1: Ala Arg Leu Gly Ala Arg Pro Cys Gly Leu ArgGlu Leu Glu Val Arg 1 5 10 15 Val Ser Glu Leu Gly Leu Gly Tyr Ala SerAsp Glu Thr Val Leu Phe 20 25 30 Arg Tyr Cys Ala Gly Ala Cys Glu Ala AlaAla Arg Val Tyr Asp Leu 35 40 45 Gly Leu Arg Arg Leu Arg Gln Arg Arg ArgLeu Arg Arg Glu Arg Val 50 55 60 Arg Ala Gln Pro Cys Cys Arg Pro Thr AlaTyr Glu Asp Glu Val Ser 65 70 75 80 Phe Leu Asp Ala His Ser Arg Tyr HisThr Val His Glu Leu Ser Ala 85 90 95 Arg Glu Cys Ala Cys Val 100 (2)INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:100 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: unknown (D)TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 2: Pro Gly Ala Arg Pro Cys Gly Leu Arg Glu Leu Glu Val ArgVal Ser 1 5 10 15 Glu Leu Gly Leu Gly Tyr Thr Ser Asp Glu Thr Val LeuPhe Arg Tyr 20 25 30 Cys Ala Gly Ala Cys Glu Ala Ala Ile Arg Ile Tyr AspLeu Gly Leu 35 40 45 Arg Arg Leu Arg Gln Arg Arg Arg Val Arg Arg Glu ArgAla Arg Ala 50 55 60 His Pro Cys Cys Arg Pro Thr Ala Tyr Glu Asp Glu ValSer Phe Leu 65 70 75 80 Asp Val His Ser Arg Tyr His Thr Leu Gln Glu LeuSer Ala Arg Glu 85 90 95 Cys Ala Cys Val 100 (2) INFORMATION FOR SEQ IDNO: 3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 16 amino acids (B)TYPE: amino acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: linear (ii)MOLECULE TYPE: peptide (ix) FEATURE: (A) NAME/KEY: Modified-site (B)LOCATION: 6 (D) OTHER INFORMATION: /note= “ANY AMINO ACID” (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 3: Ser Gly Ala Arg Pro Xaa Gly Leu Arg Glu LeuGlu Val Ser Val Ser 1 5 10 15 (2) INFORMATION FOR SEQ ID NO: 4: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 10 amino acids (B) TYPE: aminoacid (C) STRANDEDNESS: unknown (D) TOPOLOGY: linear (ii) MOLECULE TYPE:peptide (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 1 (D)OTHER INFORMATION: /note= “ANY AMINO ACID” (ix) FEATURE: (A) NAME/KEY:Modified-site (B) LOCATION: 6 (D) OTHER INFORMATION: /note= “SERINE ORCYSTEINE” (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: Xaa Cys Ala Gly AlaXaa Glu Ala Ala Val 1 5 10 (2) INFORMATION FOR SEQ ID NO: 5: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 amino acids (B) TYPE: aminoacid (C) STRANDEDNESS: unknown (D) TOPOLOGY: linear (ii) MOLECULE TYPE:peptide (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 1 (D)OTHER INFORMATION: /note= “ANY AMINO ACID” (ix) FEATURE: (A) NAME/KEY:Modified-site (B) LOCATION: 2 (D) OTHER INFORMATION: /note= “ANY AMINOACID” (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 17 (D)OTHER INFORMATION: /note= “GLUTAMINE OR GLUTAMIC ACID” (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 5: Xaa Xaa Val Glu Ala Lys Pro Cys Cys Gly ProThr Ala Tyr Glu Asp 1 5 10 15 Xaa Val Ser Phe Leu Ser Val 20 (2)INFORMATION FOR SEQ ID NO: 6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:10 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: unknown (D)TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 6: Tyr His Thr Leu Gln Glu Leu Ser Ala Arg 1 5 10 (2)INFORMATION FOR SEQ ID NO: 7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:197 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: unknown (D)TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 7: Met Gln Arg Trp Lys Ala Ala Ala Leu Ala Ser Val Leu CysSer Ser 1 5 10 15 Val Leu Ser Ile Trp Met Cys Arg Glu Gly Leu Leu LeuSer His Arg 20 25 30 Leu Gly Pro Ala Leu Val Pro Leu His Arg Leu Pro ArgThr Leu Asp 35 40 45 Ala Arg Ile Ala Arg Leu Ala Gln Tyr Arg Ala Leu LeuGln Gly Ala 50 55 60 Pro Asp Ala Met Glu Leu Arg Glu Leu Thr Pro Trp AlaGly Arg Pro 65 70 75 80 Pro Gly Pro Arg Arg Arg Ala Gly Pro Arg Arg ArgArg Ala Arg Ala 85 90 95 Arg Leu Gly Ala Arg Pro Cys Gly Leu Arg Glu LeuGlu Val Arg Val 100 105 110 Ser Glu Leu Gly Leu Gly Tyr Ala Ser Asp GluThr Val Leu Phe Arg 115 120 125 Tyr Cys Ala Gly Ala Cys Glu Ala Ala AlaArg Val Tyr Asp Leu Gly 130 135 140 Leu Arg Arg Leu Arg Gln Arg Arg ArgLeu Arg Arg Glu Arg Val Arg 145 150 155 160 Ala Gln Pro Cys Cys Arg ProThr Ala Tyr Glu Asp Glu Val Ser Phe 165 170 175 Leu Asp Ala His Ser ArgTyr His Thr Val His Glu Leu Ser Ala Arg 180 185 190 Glu Cys Ala Cys Val195 (2) INFORMATION FOR SEQ ID NO: 8: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 195 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: unknown(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 8: Met Arg Arg Trp Lys Ala Ala Ala Leu Val SerLeu Ile Cys Ser Ser 1 5 10 15 Leu Leu Ser Val Trp Met Cys Gln Glu GlyLeu Leu Leu Gly His Arg 20 25 30 Leu Gly Pro Ala Leu Ala Pro Leu Arg ArgPro Pro Arg Thr Leu Asp 35 40 45 Ala Arg Ile Ala Arg Leu Ala Gln Tyr ArgAla Leu Leu Gln Gly Ala 50 55 60 Pro Asp Ala Val Glu Leu Arg Glu Leu SerPro Trp Ala Ala Arg Ile 65 70 75 80 Pro Gly Pro Arg Arg Arg Ala Gly ProArg Arg Arg Arg Ala Arg Pro 85 90 95 Gly Ala Arg Pro Cys Gly Leu Arg GluLeu Glu Val Arg Val Ser Glu 100 105 110 Leu Gly Leu Gly Tyr Thr Ser AspGlu Thr Val Leu Phe Arg Tyr Cys 115 120 125 Ala Gly Ala Cys Glu Ala AlaIle Arg Ile Tyr Asp Leu Gly Leu Arg 130 135 140 Arg Leu Arg Gln Arg ArgArg Val Arg Arg Glu Arg Ala Arg Ala His 145 150 155 160 Pro Cys Cys ArgPro Thr Ala Tyr Glu Asp Glu Val Ser Phe Leu Asp 165 170 175 Val His SerArg Tyr His Thr Leu Gln Glu Leu Ser Ala Arg Glu Cys 180 185 190 Ala CysVal 195 (2) INFORMATION FOR SEQ ID NO: 9: (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 306 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 9: GCGCGGTTGG GGGCGCGGCC TTGCGGGCTG CGCGAGCTGGAGGTGCGCGT GAGCGAGCTG 60 GGCCTGGGCT ACGCGTCCGA CGAGACGGTG CTGTTCCGCTACTGCGCAGG CGCCTGCGAG 120 GCTGCCGCGC GCGTCTACGA CCTCGGGCTG CGACGACTGCGCCAGCGGCG GCGCCTGCGG 180 CGGGAGCGGG TGCGCGCGCA GCCCTGCTGC CGCCCGACGGCCTACGAGGA CGAGGTGTCC 240 TTCCTGGACG CGCACAGCCG CTACCACACG GTGCACGAGCTGTCGGCGCG CGAGTGCGCC 300 TGCGTG 306 (2) INFORMATION FOR SEQ ID NO: 10:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 300 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: CCGGGGGCTCGGCCTTGTGG GCTGCGCGAG CTCGAGGTGC GCGTGAGCGA GCTGGGCCTG 60 GGCTACACGTCGGATGAGAC CGTGCTGTTC CGCTACTGCG CAGGCGCGTG CGAGGCGGCC 120 ATCCGCATCTACGACCTGGG CCTTCGGCGC CTGCGCCAGC GGAGGCGCGT GCGCAGAGAG 180 CGGGCGCGGGCGCACCCGTG TTGTCGCCCG ACGGCCTATG AGGACGAGGT GTCCTTCCTG 240 GACGTGCACAGCCGCTACCA CACGCTGCAA GAGCTGTCGG CGCGGGAGTG CGCGTGCGTG 300 (2)INFORMATION FOR SEQ ID NO: 11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:591 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQID NO: 11: ATGCAGCGCT GGAAGGCGGC GGCCTTGGCC TCAGTGCTCT GCAGCTCCGTGCTGTCCATC 60 TGGATGTGTC GAGAGGGCCT GCTTCTCAGC CACCGCCTCG GACCTGCGCTGGTCCCCCTG 120 CACCGCCTGC CTCGAACCCT GGACGCCCGG ATTGCCCGCC TGGCCCAGTACCGTGCACTC 180 CTGCAGGGGG CCCCGGATGC GATGGAGCTG CGCGAGCTGA CGCCCTGGGCTGGGCGGCCC 240 CCAGGTCCGC GCCGTCGGGC GGGGCCCCGG CGGCGGCGCG CGCGTGCGCGGTTGGGGGCG 300 CGGCCTTGCG GGCTGCGCGA GCTGGAGGTG CGCGTGAGCG AGCTGGGCCTGGGCTACGCG 360 TCCGACGAGA CGGTGCTGTT CCGCTACTGC GCAGGCGCCT GCGAGGCTGCCGCGCGCGTC 420 TACGACCTCG GGCTGCGACG ACTGCGCCAG CGGCGGCGCC TGCGGCGGGAGCGGGTGCGC 480 GCGCAGCCCT GCTGCCGCCC GACGGCCTAC GAGGACGAGG TGTCCTTCCTGGACGCGCAC 540 AGCCGCTACC ACACGGTGCA CGAGCTGTCG GCGCGCGAGT GCGCCTGCGT G591 (2) INFORMATION FOR SEQ ID NO: 12: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 585 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 12: ATGAGGCGCT GGAAGGCAGC GGCCCTGGTG TCGCTCATCT GCAGCTCCCTGCTATCTGTC 60 TGGATGTGCC AGGAGGGTCT GCTCTTGGGC CACCGCCTGG GACCCGCGCTTGCCCCGCTA 120 CGACGCCCTC CACGCACCCT GGACGCCCGC ATCGCCCGCC TGGCCCAGTATCGCGCTCTG 180 CTCCAGGGCG CCCCCGACGC GGTGGAGCTT CGAGAACTTT CTCCCTGGGCTGCCCGCATC 240 CCGGGACCGC GCCGTCGAGC GGGTCCCCGG CGTCGGCGGG CGCGGCCGGGGGCTCGGCCT 300 TGTGGGCTGC GCGAGCTCGA GGTGCGCGTG AGCGAGCTGG GCCTGGGCTACACGTCGGAT 360 GAGACCGTGC TGTTCCGCTA CTGCGCAGGC GCGTGCGAGG CGGCCATCCGCATCTACGAC 420 CTGGGCCTTC GGCGCCTGCG CCAGCGGAGG CGCGTGCGCA GAGAGCGGGCGCGGGCGCAC 480 CCGTGTTGTC GCCCGACGGC CTATGAGGAC GAGGTGTCCT TCCTGGACGTGCACAGCCGC 540 TACCACACGC TGCAAGAGCT GTCGGCGCGG GAGTGCGCGT GCGTG 585 (2)INFORMATION FOR SEQ ID NO: 13: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:348 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQID NO: 13: GGAGGGAGAG CGCGCGGTGG TTTCGTCCGT GTGCCCCGCG CCCGGCGCTCCTCGCGTGGC 60 CCCGCGTCCT GAGCGCGCTC CAGCCTCCCA CGCGCGCCAC CCCGGGGTTCACTGAGCCCG 120 GCGAGCCCGG GGAAGACAGA GAAAGAGAGG CCAGGGGGGG AACCCCATGGCCCGGCCCGT 180 GTCCCGCACC CTGTGCGGTG GCCTCCTCCG GCACGGGGTC CCCGGGTCGCCTCCGGTCCC 240 CGCGATCCGG ATGGCGCACG CAGTGGCTGG GGCCGGGCCG GGCTCGGGTGGTCGGAGGAG 300 TCACCACTGA CCGGGTCATC TGGAGCCCGT GGCAGGCCGA GGCCCAGG 348(2) INFORMATION FOR SEQ ID NO: 14: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 87 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 14: TGCTACCTCA CGCCCCCCGA CCTGCGAAAG GGCCCTCCCT GCCGACCCTCGCTGAGAACT 60 GACTTCACAT AAAGTGTGGG AACTCCC 87 (2) INFORMATION FOR SEQID NO: 15: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 amino acids (B)TYPE: amino acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: linear (ii)MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15: Met GlnArg Trp Lys Ala Ala Ala Leu Ala Ser Val Leu Cys Ser Ser 1 5 10 15 ValLeu Ser (2) INFORMATION FOR SEQ ID NO: 16: (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 19 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:unknown (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 16: Met Arg Arg Trp Lys Ala Ala Ala Leu Val SerLeu Ile Cys Ser Ser 1 5 10 15 Leu Leu Ser (2) INFORMATION FOR SEQ ID NO:17: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 57 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17: ATGCAGCGCTGGAAGGCGGC GGCCTTGGCC TCAGTGCTCT GCAGCTCCGT GCTGTCC 57 (2) INFORMATIONFOR SEQ ID NO: 18: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 57 basepairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: ATGAGGCGCT GGAAGGCAGC GGCCCTGGTG TCGCTCATCT GCAGCTCCCT GCTATCT 57(2) INFORMATION FOR SEQ ID NO: 19: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 76 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: unknown(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 19: Ile Trp Met Cys Arg Glu Gly Leu Leu Leu SerHis Arg Leu Gly Pro 1 5 10 15 Ala Leu Val Pro Leu His Arg Leu Pro ArgThr Leu Asp Ala Arg Ile 20 25 30 Ala Arg Leu Ala Gln Tyr Arg Ala Leu LeuGln Gly Ala Pro Asp Ala 35 40 45 Met Glu Leu Arg Glu Leu Thr Pro Trp AlaGly Arg Pro Pro Gly Pro 50 55 60 Arg Arg Arg Ala Gly Pro Arg Arg Arg ArgAla Arg 65 70 75 (2) INFORMATION FOR SEQ ID NO: 20: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 228 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 20: ATCTGGATGT GTCGAGAGGG CCTGCTTCTCAGCCACCGCC TCGGACCTGC GCTGGTCCCC 60 CTGCACCGCC TGCCTCGAAC CCTGGACGCCCGGATTGCCC GCCTGGCCCA GTACCGTGCA 120 CTCCTGCAGG GGGCCCCGGA TGCGATGGAGCTGCGCGAGC TGACGCCCTG GGCTGGGCGG 180 CCCCCAGGTC CGCGCCGTCG GGCGGGGCCCCGGCGGCGGC GCGCGCGT 228 (2) INFORMATION FOR SEQ ID NO: 21: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 228 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 21: GTCTGGATGT GCCAGGAGGG TCTGCTCTTGGGCCACCGCC TGGGACCCGC GCTTGCCCCG 60 CTACGACGCC CTCCACGCAC CCTGGACGCCCGCATCGCCC GCCTGGCCCA GTATCGCGCT 120 CTGCTCCAGG GCGCCCCCGA CGCGGTGGAGCTTCGAGAAC TTTCTCCCTG GGCTGCCCGC 180 ATCCCGGGAC CGCGCCGTCG AGCGGGTCCCCGGCGTCGGC GGGCGCGG 228 (2) INFORMATION FOR SEQ ID NO: 22: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 76 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: unknown (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22: Val Trp Met Cys Gln Glu GlyLeu Leu Leu Gly His Arg Leu Gly Pro 1 5 10 15 Ala Leu Ala Pro Leu ArgArg Pro Pro Arg Thr Leu Asp Ala Arg Ile 20 25 30 Ala Arg Leu Ala Gln TyrArg Ala Leu Leu Gln Gly Ala Pro Asp Ala 35 40 45 Val Glu Leu Arg Glu LeuSer Pro Trp Ala Ala Arg Ile Pro Gly Pro 50 55 60 Arg Arg Arg Ala Gly ProArg Arg Arg Arg Ala Arg 65 70 75 (2) INFORMATION FOR SEQ ID NO: 23: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 95 amino acids (B) TYPE: aminoacid (C) STRANDEDNESS: unknown (D) TOPOLOGY: linear (ii) MOLECULE TYPE:peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23: Met Gln Arg Trp LysAla Ala Ala Leu Ala Ser Val Leu Cys Ser Ser 1 5 10 15 Val Leu Ser IleTrp Met Cys Arg Glu Gly Leu Leu Leu Ser His Arg 20 25 30 Leu Gly Pro AlaLeu Val Pro Leu His Arg Leu Pro Arg Thr Leu Asp 35 40 45 Ala Arg Ile AlaArg Leu Ala Gln Tyr Arg Ala Leu Leu Gln Gly Ala 50 55 60 Pro Asp Ala MetGlu Leu Arg Glu Leu Thr Pro Trp Ala Gly Arg Pro 65 70 75 80 Pro Gly ProArg Arg Arg Ala Gly Pro Arg Arg Arg Arg Ala Arg 85 90 95 (2) INFORMATIONFOR SEQ ID NO: 24: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 95 aminoacids (B) TYPE: amino acid (C) STRANDEDNESS: unknown (D) TOPOLOGY:linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: Met Arg Arg Trp Lys Ala Ala Ala Leu Val Ser Leu Ile Cys Ser Ser 1 510 15 Leu Leu Ser Val Trp Met Cys Gln Glu Gly Leu Leu Leu Gly His Arg 2025 30 Leu Gly Pro Ala Leu Ala Pro Leu Arg Arg Pro Pro Arg Thr Leu Asp 3540 45 Ala Arg Ile Ala Arg Leu Ala Gln Tyr Arg Ala Leu Leu Gln Gly Ala 5055 60 Pro Asp Ala Val Glu Leu Arg Glu Leu Ser Pro Trp Ala Ala Arg Ile 6570 75 80 Pro Gly Pro Arg Arg Arg Ala Gly Pro Arg Arg Arg Arg Ala Arg 8590 95 (2) INFORMATION FOR SEQ ID NO: 25: (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 285 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 25: ATGCAGCGCT GGAAGGCGGC GGCCTTGGCC TCAGTGCTCTGCAGCTCCGT GCTGTCCATC 60 TGGATGTGTC GAGAGGGCCT GCTTCTCAGC CACCGCCTCGGACCTGCGCT GGTCCCCCTG 120 CACCGCCTGC CTCGAACCCT GGACGCCCGG ATTGCCCGCCTGGCCCAGTA CCGTGCACTC 180 CTGCAGGGGG CCCCGGATGC GATGGAGCTG CGCGAGCTGACGCCCTGGGC TGGGCGGCCC 240 CCAGGTCCGC GCCGTCGGGC GGGGCCCCGG CGGCGGCGCGCGCGT 285 (2) INFORMATION FOR SEQ ID NO: 26: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 285 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 26: ATGAGGCGCT GGAAGGCAGC GGCCCTGGTGTCGCTCATCT GCAGCTCCCT GCTATCTGTC 60 TGGATGTGCC AGGAGGGTCT GCTCTTGGGCCACCGCCTGG GACCCGCGCT TGCCCCGCTA 120 CGACGCCCTC CACGCACCCT GGACGCCCGCATCGCCCGCC TGGCCCAGTA TCGCGCTCTG 180 CTCCAGGGCG CCCCCGACGC GGTGGAGCTTCGAGAACTTT CTCCCTGGGC TGCCCGCATC 240 CCGGGACCGC GCCGTCGAGC GGGTCCCCGGCGTCGGCGGG CGCGG 285 (2) INFORMATION FOR SEQ ID NO: 27: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 169 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 27: ATGCAGCGCT GGAAGGCGGC GGCCTTGGCCTCAGTGCTCT GCAGCTCCGT GCTGTCCATC 60 TGGATGTGTC GAGAGGGCCT GCTTCTCAGCCACCGCCTCG GACCTGCGCT GGTCCCCCTG 120 CACCGCCTGC CTCGAACCCT GGACGCCCGGATTGCCCGCC TGGCCCAGT 169 (2) INFORMATION FOR SEQ ID NO: 28: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 425 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 28: ACCGTGCACT CCTGCAGGGG GCCCCGGATGCGATGGAGCT GCGCGAGCTG ACGCCCTGGG 60 CTGGGCGGCC CCCAGGTCCG CGCCGTCGGGCGGGGCCCCG GCGGCGGCGC GCGCGTGCGC 120 GGTTGGGGGC GCGGCCTTGC GGGCTGCGCGAGCTGGAGGT GCGCGTGAGC GAGCTGGGCC 180 TGGGCTACGC GTCCGACGAG ACGGTGCTGTTCCGCTACTG CGCAGGCGCC TGCGAGGCTG 240 CCGCGCGCGT CTACGACCTC GGGCTGCGACGACTGCGCCA GCGGCGGCGC CTGCGGCGGG 300 AGCGGGTGCG CGCGCAGCCC TGCTGCCGCCCGACGGCCTA CGAGGACGAG GTGTCCTTCC 360 TGGACGCGCA CAGCCGCTAC CACACGGTGCACGAGCTGTC GGCGCGCGAG TGCGCCTGCG 420 TGTGA 425 (2) INFORMATION FOR SEQID NO: 29: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 169 base pairs (B)TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29: ATGAGGCGCTGGAAGGCAGC GGCCCTGGTG TCGCTCATCT GCAGCTCCCT GCTATCTGTC 60 TGGATGTGCCAGGAGGGTCT GCTCTTGGGC CACCGCCTGG GACCCGCGCT TGCCCCGCTA 120 CGACGCCCTCCACGCACCCT GGACGCCCGC ATCGCCCGCC TGGCCCAGT 169 (2) INFORMATION FOR SEQID NO: 30: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 419 base pairs (B)TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30: ATCGCGCTCTGCTCCAGGGC GCCCCCGACG CGGTGGAGCT TCGAGAACTT TCTCCCTGGG 60 CTGCCCGCATCCCGGGACCG CGCCGTCGAG CGGGTCCCCG GCGTCGGCGG GCGCGGCCGG 120 GGGCTCGGCCTTGTGGGCTG CGCGAGCTCG AGGTGCGCGT GAGCGAGCTG GGCCTGGGCT 180 ACACGTCGGATGAGACCGTG CTGTTCCGCT ACTGCGCAGG CGCGTGCGAG GCGGCCATCC 240 GCATCTACGACCTGGGCCTT CGGCGCCTGC GCCAGCGGAG GCGCGTGCGC AGAGAGCGGG 300 CGCGGGCGCACCCGTGTTGT CGCCCGACGG CCTATGAGGA CGAGGTGTCC TTCCTGGACG 360 TGCACAGCCGCTACCACACG CTGCAAGAGC TGTCGGCGCG GGAGTGCGCG TGCGTGTGA 419 (2)INFORMATION FOR SEQ ID NO: 31: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:94 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: unknown (D)TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 31: Cys Gly Leu Arg Glu Leu Glu Val Arg Val Ser Glu Leu GlyLeu Gly 1 5 10 15 Tyr Ala Ser Asp Glu Thr Val Leu Phe Arg Tyr Cys AlaGly Ala Cys 20 25 30 Glu Ala Ala Ala Arg Val Tyr Asp Leu Gly Leu Arg ArgLeu Arg Gln 35 40 45 Arg Arg Arg Leu Arg Arg Glu Arg Val Arg Ala Gln ProCys Cys Arg 50 55 60 Pro Thr Ala Tyr Glu Asp Glu Val Ser Phe Leu Asp AlaHis Ser Arg 65 70 75 80 Tyr His Thr Val His Glu Leu Ser Ala Arg Glu CysAla Cys 85 90 (2) INFORMATION FOR SEQ ID NO: 32: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 94 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: unknown (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32: Cys Gly Leu Arg Glu Leu GluVal Arg Val Ser Glu Leu Gly Leu Gly 1 5 10 15 Tyr Thr Ser Asp Glu ThrVal Leu Phe Arg Tyr Cys Ala Gly Ala Cys 20 25 30 Glu Ala Ala Ile Arg IleTyr Asp Leu Gly Leu Arg Arg Leu Arg Gln 35 40 45 Arg Arg Arg Val Arg ArgGlu Arg Ala Arg Ala His Pro Cys Cys Arg 50 55 60 Pro Thr Ala Tyr Glu AspGlu Val Ser Phe Leu Asp Val His Ser Arg 65 70 75 80 Tyr His Thr Leu GlnGlu Leu Ser Ala Arg Glu Cys Ala Cys 85 90 (2) INFORMATION FOR SEQ ID NO:33: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE:amino acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: linear (ii) MOLECULETYPE: peptide (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 2(D) OTHER INFORMATION: /note= “SERINE OR THREONINE” (ix) FEATURE: (A)NAME/KEY: Modified-site (B) LOCATION: 3 (D) OTHER INFORMATION: /note=“GLUTAMIC ACID OR ASPARTIC ACID” (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33: Val Xaa Xaa Leu Gly Leu Gly Tyr 1 5 (2) INFORMATION FOR SEQ ID NO:34: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 amino acids (B) TYPE:amino acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: linear (ii) MOLECULETYPE: peptide (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 2(D) OTHER INFORMATION: /note= “THREONINE OR GLUTAMIC ACID” (ix) FEATURE:(A) NAME/KEY: Modified-site (B) LOCATION: 3 (D) OTHER INFORMATION:/note= “VALINE OR LEUCINE” (ix) FEATURE: (A) NAME/KEY: Modified-site (B)LOCATION: 4 (D) OTHER INFORMATION: /note= “LEUCINE OR ISOLEUCINE” (ix)FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 9 (D) OTHERINFORMATION: /note= “ALANINE OR SERINE” (ix) FEATURE: (A) NAME/KEY:Modified-site (B) LOCATION: 11 (D) OTHER INFORMATION: /note= “ALANINE ORSERINE” (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 13 (D)OTHER INFORMATION: /note= “GLUTAMIC ACID OR ASPARTIC ACID” (ix) FEATURE:(A) NAME/KEY: Modified-site (B) LOCATION: 14 (D) OTHER INFORMATION:/note= “ALANINE OR SERINE” (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34: GluXaa Xaa Xaa Phe Arg Tyr Cys Xaa Gly Xaa Cys Xaa Xaa Ala 1 5 10 15 (2)INFORMATION FOR SEQ ID NO: 35: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:15 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: unknown (D)TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: (A) NAME/KEY:Modified-site (B) LOCATION: 5 (D) OTHER INFORMATION: /note= “THREONINEOR VALINE OR ISOLEUCINE” (ix) FEATURE: (A) NAME/KEY: Modified-site (B)LOCATION: 7 (D) OTHER INFORMATION: /note= “TYROSINE OR PHENYLALANINE”(ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 8 (D) OTHERINFORMATION: /note= “GLUTAMIC ACID OR ASPARTIC ACID” (ix) FEATURE: (A)NAME/KEY: Modified-site (B) LOCATION: 10 (D) OTHER INFORMATION: /note=“GLUTAMIC ACID OR ASPARTIC ACID” (ix) FEATURE: (A) NAME/KEY:Modified-site (B) LOCATION: 11 (D) OTHER INFORMATION: /note= “VALINE ORLEUCINE” (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 35: Cys Cys Arg Pro XaaAla Xaa Xaa Asp Xaa Xaa Ser Phe Leu Asp 1 5 10 15 (2) INFORMATION FORSEQ ID NO: 36: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 11 amino acids(B) TYPE: amino acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: linear (ii)MOLECULE TYPE: peptide (ix) FEATURE: (A) NAME/KEY: Modified-site (B)LOCATION: 5 (D) OTHER INFORMATION: /note= “ALANINE OR SERINE” (ix)FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 7 (D) OTHERINFORMATION: /note= “ALANINE OR SERINE” (ix) FEATURE: (A) NAME/KEY:Modified-site (B) LOCATION: 9 (D) OTHER INFORMATION: /note= “GLUTAMICACID OR ASPARTIC ACID” (ix) FEATURE: (A) NAME/KEY: Modified-site (B)LOCATION: 10 (D) OTHER INFORMATION: /note= “SERINE OR ALANINE” (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 36: Phe Arg Tyr Cys Xaa Gly Xaa Cys XaaXaa Ala 1 5 10 (2) INFORMATION FOR SEQ ID NO: 37: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 11 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: unknown (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide(ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 5 (D) OTHERINFORMATION: /note= “ALANINE OR SERINE” (ix) FEATURE: (A) NAME/KEY:Modified-site (B) LOCATION: 7 (D) OTHER INFORMATION: /note= “ALANINE ORSERINE” (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 9 (D)OTHER INFORMATION: /note= “GLUTAMIC ACID OR ASPARTIC ACID” (ix) FEATURE:(A) NAME/KEY: Modified-site (B) LOCATION: 10 (D) OTHER INFORMATION:/note= “SERINE OR ALANINE” (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37: PheArg Tyr Cys Xaa Gly Xaa Cys Xaa Xaa Ala 1 5 10 (2) INFORMATION FOR SEQID NO: 38: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 10 amino acids (B)TYPE: amino acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: linear (ii)MOLECULE TYPE: peptide (ix) FEATURE: (A) NAME/KEY: Modified-site (B)LOCATION: 5 (D) OTHER INFORMATION: /note= “ISOLEUCINE OR THREONINE ORVALINE” (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 7 (D)OTHER INFORMATION: /note= “TYROSINE OR PHENYLALANINE” (ix) FEATURE: (A)NAME/KEY: Modified-site (B) LOCATION: 8 (D) OTHER INFORMATION: /note=“GLUTAMIC ACID OR ASPARTIC ACID” (ix) FEATURE: (A) NAME/KEY:Modified-site (B) LOCATION: 10 (D) OTHER INFORMATION: /note= “GLUTAMICACID OR ASPARTIC ACID” (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 38: Cys CysArg Pro Xaa Ala Xaa Xaa Asp Xaa 1 5 10 (2) INFORMATION FOR SEQ ID NO:39: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 10 amino acids (B) TYPE:amino acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: linear (ii) MOLECULETYPE: peptide (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 2(D) OTHER INFORMATION: /note= “TYROSINE OR PHENYLALANINE” (ix) FEATURE:(A) NAME/KEY: Modified-site (B) LOCATION: 3 (D) OTHER INFORMATION:/note= “GLUTAMIC ACID OR ASPARTIC ACID” (ix) FEATURE: (A) NAME/KEY:Modified-site (B) LOCATION: 5 (D) OTHER INFORMATION: /note= “GLUTAMICACID OR ASPARTIC ACID” (ix) FEATURE: (A) NAME/KEY: Modified-site (B)LOCATION: 6 (D) OTHER INFORMATION: /note= “VALINE OR LEUCINE” (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 39: Ala Xaa Xaa Asp Xaa Xaa Ser Phe LeuAsp 1 5 10 (2) INFORMATION FOR SEQ ID NO: 40: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: unknown (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide(ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 2 (D) OTHERINFORMATION: /note= “GLUTAMIC ACID OR THREONINE” (ix) FEATURE: (A)NAME/KEY: Modified-site (B) LOCATION: 3 (D) OTHER INFORMATION: /note=“LEUCINE OR VALINE” (ix) FEATURE: (A) NAME/KEY: Modified-site (B)LOCATION: 4 (D) OTHER INFORMATION: /note= “ISOLEUCINE OR LEUCINE” (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 40: Glu Xaa Xaa Xaa Phe Arg Tyr Cys 1 5(2) INFORMATION FOR SEQ ID NO: 41: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 13 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: unknown(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: (A)NAME/KEY: Modified-site (B) LOCATION: 2 (D) OTHER INFORMATION: /note=“GLUTAMIC ACID OR THREONINE” (ix) FEATURE: (A) NAME/KEY: Modified-site(B) LOCATION: 3 (D) OTHER INFORMATION: /note= “LEUCINE OR VALINE” (ix)FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 4 (D) OTHERINFORMATION: /note= “ISOLEUCINE OR LEUCINE” (ix) FEATURE: (A) NAME/KEY:Modified-site (B) LOCATION: 9 (D) OTHER INFORMATION: /note= “SERINE ORALANINE” (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 11 (D)OTHER INFORMATION: /note= “SERINE OR ALANINE” (ix) FEATURE: (A)NAME/KEY: Modified-site (B) LOCATION: 13 (D) OTHER INFORMATION: /note=“GLUTAMIC ACID OR ASPARTIC ACID” (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41: Glu Xaa Xaa Xaa Phe Arg Tyr Cys Xaa Gly Xaa Cys Xaa 1 5 10 (2)INFORMATION FOR SEQ ID NO: 42: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:23 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQID NO: 42: GTNWSNGANY TNGGNYTNGG NTA 23 (2) INFORMATION FOR SEQ ID NO:43: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 32 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 43: TTYMGNTAYTGYDSNGGNDS NTGYGANKCN GC 32 (2) INFORMATION FOR SEQ ID NO: 44: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 32 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 44: GCNGMNTCRC ANSHNCCNSHRCARTANCKR AA 32 (2) INFORMATION FOR SEQ ID NO: 45: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 29 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 45: TCRTCNTCRW ANGCNRYNGG NCKRCARCA 29(2) INFORMATION FOR SEQ ID NO: 46: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 29 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 46: TCNARRAANS WNAVNTCRTC NTCRWANGC 29 (2) INFORMATION FORSEQ ID NO: 47: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 base pairs(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 47:GARRMNBTNH TNTTYMGNTA YTG 23 (2) INFORMATION FOR SEQ ID NO: 48: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 38 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 48: GARRMNBTNH TNTTYMGNTAYTGYDSNGGN DSNTGHGA 38 (2) INFORMATION FOR SEQ ID NO: 49: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 16 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: unknown (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 49: Ser Gly Ala Arg Pro Xaa GlyLeu Arg Glu Leu Glu Val Ser Val Ser 1 5 10 15 (2) INFORMATION FOR SEQ IDNO: 50: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 17 base pairs (B)TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 50: CCNACNGCNTAYGARGA 17 (2) INFORMATION FOR SEQ ID NO: 51: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 20 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: unknown (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 51: Ala Arg Ala His Pro Cys CysArg Pro Thr Ala Tyr Glu Asp Glu Val 1 5 10 15 Ser Phe Leu Asp 20 (2)INFORMATION FOR SEQ ID NO: 52: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQID NO: 52: ARYTCYTGNA RNGTRTGRTA 20 (2) INFORMATION FOR SEQ ID NO: 53:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 28 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 53: GACGAGGTGTCCTTCCTGGA CGTACACA 28 (2) INFORMATION FOR SEQ ID NO: 54: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 34 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 54: TAGCGGCTGT GTACGTCCAG GAAGGACACCTCGT 34 (2) INFORMATION FOR SEQ ID NO: 55: (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 55: CAGCGACGAC GCGTGCGCAA AGAGCG 26 (2)INFORMATION FOR SEQ ID NO: 56: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:47 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQID NO: 56: TAYGARGACG AGGTGTCCTT CCTGGACGTA CACAGCCGCT AYCAYAC 47 (2)INFORMATION FOR SEQ ID NO: 57: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:26 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQID NO: 57: GCGGCCATCC GCATCTACGA CCTGGG 26 (2) INFORMATION FOR SEQ IDNO: 58: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 base pairs (B)TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 58: CRTAGGCCGTCGGGCGRCAR CACGGGT 27 (2) INFORMATION FOR SEQ ID NO: 59: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 59: GCGCCGAAGG CCCAGGTCGT AGATGCG 27(2) INFORMATION FOR SEQ ID NO: 60: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 29 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 60: CGCTACTGCG CAGGCGCGTG CGARGCGGC 29 (2) INFORMATION FORSEQ ID NO: 61: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 base pairs(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 61:CGCCGACAGC TCTTGCAGCG TRTGGTA 27 (2) INFORMATION FOR SEQ ID NO: 62: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 62: GAGCTGGGCC TGGGCTACGCGTCCGACGAG 30 (2) INFORMATION FOR SEQ ID NO: 63: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 39 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 63: GCGACGCGTA CCATGAGGCG CTGGAAGGCAGCGGCCCTG 39 (2) INFORMATION FOR SEQ ID NO: 64: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 64: GACGGATCCG CATCACACGC ACGCGCACTC 30(2) INFORMATION FOR SEQ ID NO: 65: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 29 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 65: GACCATATGC CGGGGGCTCG GCCTTGTGG 29 (2) INFORMATION FORSEQ ID NO: 66: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 66:GACGGATCCG CATCACACGC ACGCGCACTC 30 (2) INFORMATION FOR SEQ ID NO: 67:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 26 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 67: CAGCGACGACGCGTGCGCAA AGAGCG 26 (2) INFORMATION FOR SEQ ID NO: 68: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 34 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 68: TAGCGGCTGT GTACGTCCAG GAAGGACACCTCGT 34 (2) INFORMATION FOR SEQ ID NO: 69: (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 69: AAAAATCGGG GGTGYGTCTT A 21 (2) INFORMATIONFOR SEQ ID NO: 70: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 basepairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:70: CATGCCTGGC CTACYTTGTC A 21 (2) INFORMATION FOR SEQ ID NO: 71: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 71: CTGGCGTCCC AMCAAGGGTCTTCG 24 (2) INFORMATION FOR SEQ ID NO: 72: (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 23 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 72: GCCAGTGGTG CCGTCGAGGC GGG 23 (2) INFORMATIONFOR SEQ ID NO: 73: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 basepairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:73: GGCCCAGGAT GAGGCGCTGG AAGG 24 (2) INFORMATION FOR SEQ ID NO: 74: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 74: CCACTCCACT GCCTGAWATTCWACCCC 27 (2) INFORMATION FOR SEQ ID NO: 75: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 75: CCATGTGATT ATCGACCATT CGGC 24 (2)INFORMATION FOR SEQ ID NO: 76: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:134 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: unknown (D)TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 76: Ser Pro Asp Lys Gln Met Ala Val Leu Pro Arg Arg Glu ArgAsn Arg 1 5 10 15 Gln Ala Ala Ala Ala Asn Pro Glu Asn Ser Arg Gly LysGly Arg Arg 20 25 30 Gly Gln Arg Gly Lys Asn Arg Gly Cys Val Leu Thr AlaIle His Leu 35 40 45 Asn Val Thr Asp Leu Gly Leu Gly Tyr Glu Thr Lys GluGlu Leu Ile 50 55 60 Phe Arg Tyr Cys Ser Gly Ser Cys Asp Ala Ala Glu ThrThr Tyr Asp 65 70 75 80 Lys Ile Leu Lys Asn Leu Ser Arg Asn Arg Arg LeuVal Ser Asp Lys 85 90 95 Val Gly Gln Ala Cys Cys Arg Pro Ile Ala Phe AspAsp Asp Leu Ser 100 105 110 Phe Leu Asp Asp Asn Leu Val Tyr His Ile LeuArg Lys His Ser Ala 115 120 125 Lys Arg Cys Gly Cys Ile 130 (2)INFORMATION FOR SEQ ID NO: 77: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:134 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: unknown (D)TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 77: Ser Pro Asp Lys Gln Ala Ala Ala Leu Pro Arg Arg Glu ArgAsn Arg 1 5 10 15 Gln Ala Ala Ala Ala Ser Pro Glu Asn Ser Arg Gly LysGly Arg Arg 20 25 30 Gly Gln Arg Gly Lys Asn Arg Gly Cys Val Leu Thr AlaIle His Leu 35 40 45 Asn Val Thr Asp Leu Gly Leu Gly Tyr Glu Thr Lys GluGlu Leu Ile 50 55 60 Phe Arg Tyr Cys Ser Gly Ser Cys Glu Ser Ala Glu ThrMet Tyr Asp 65 70 75 80 Lys Ile Leu Lys Asn Leu Ser Arg Ser Arg Arg LeuThr Ser Asp Lys 85 90 95 Val Gly Gln Ala Cys Cys Arg Pro Val Ala Phe AspAsp Asp Leu Ser 100 105 110 Phe Leu Asp Asp Asn Leu Val Tyr His Ile LeuArg Lys His Ser Ala 115 120 125 Lys Arg Cys Gly Cys Ile 130 (2)INFORMATION FOR SEQ ID NO: 78: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:134 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: unknown (D)TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 78: Ser Pro Asp Lys Gln Ala Ala Ala Leu Pro Arg Arg Glu ArgAsn Arg 1 5 10 15 Gln Ala Ala Ala Ala Ser Pro Glu Asn Ser Arg Gly LysGly Arg Arg 20 25 30 Gly Gln Arg Gly Lys Asn Arg Gly Cys Val Leu Thr AlaIle His Leu 35 40 45 Asn Val Thr Asp Leu Gly Leu Gly Tyr Glu Thr Lys GluGlu Leu Ile 50 55 60 Phe Arg Tyr Cys Ser Gly Ser Cys Glu Ala Ala Glu ThrMet Tyr Asp 65 70 75 80 Lys Ile Leu Lys Asn Leu Ser Arg Ser Arg Arg LeuThr Ser Asp Lys 85 90 95 Val Gly Gln Ala Cys Cys Arg Pro Val Ala Phe AspAsp Asp Leu Ser 100 105 110 Phe Leu Asp Asp Ser Leu Val Tyr His Ile LeuArg Lys His Ser Ala 115 120 125 Lys Arg Cys Gly Cys Ile 130 (2)INFORMATION FOR SEQ ID NO: 79: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:89 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: unknown (D)TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 79: Cys Arg Leu Trp Ser Leu Thr Leu Pro Val Ala Glu Leu GlyLeu Gly 1 5 10 15 Tyr Ala Ser Glu Glu Lys Val Ile Phe Arg Tyr Cys AlaGly Ser Cys 20 25 30 Pro Gln Glu Ala Arg Thr Gln His Ser Leu Val Leu AlaArg Leu Arg 35 40 45 Gly Arg Gly Arg Ala His Gly Arg Pro Cys Cys Gln ProThr Ser Tyr 50 55 60 Ala Asp Val Thr Phe Leu Asp Asp Gln His His Trp GlnGln Leu Pro 65 70 75 80 Gln Leu Ser Ala Ala Ala Cys Gly Cys 85 (2)INFORMATION FOR SEQ ID NO: 80: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:96 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: unknown (D)TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 80: Ala Leu Ala Gly Ser Cys Arg Leu Trp Ser Leu Thr Leu ProVal Ala 1 5 10 15 Glu Leu Gly Leu Gly Tyr Ala Ser Glu Glu Lys Val IlePhe Arg Tyr 20 25 30 Cys Ala Gly Ser Cys Pro Gln Glu Ala Arg Thr Gln HisSer Leu Val 35 40 45 Leu Ala Arg Leu Arg Gly Arg Gly Arg Ala His Gly ArgPro Cys Cys 50 55 60 Gln Pro Thr Ser Tyr Ala Asp Val Thr Phe Leu Asp AspGln His His 65 70 75 80 Trp Gln Gln Leu Pro Gln Leu Ser Ala Ala Ala CysGly Cys Gly Gly 85 90 95 (2) INFORMATION FOR SEQ ID NO: 81: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 134 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: unknown (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 81: Val Arg Ile Pro Gly Gly LeuPro Thr Pro Gln Phe Leu Leu Ser Lys 1 5 10 15 Pro Ser Leu Cys Leu ThrIle Leu Leu Tyr Leu Ala Leu Gly Asn Asn 20 25 30 His Val Arg Leu Pro ArgAla Leu Ala Gly Ser Cys Arg Leu Trp Ser 35 40 45 Leu Thr Leu Pro Val AlaGlu Leu Gly Leu Gly Tyr Ala Ser Glu Glu 50 55 60 Lys Val Ile Phe Arg TyrCys Ala Gly Ser Cys Pro Gln Glu Ala Arg 65 70 75 80 Thr Gln His Ser LeuVal Leu Ala Arg Leu Arg Gly Arg Gly Arg Ala 85 90 95 His Gly Arg Pro CysCys Gln Pro Thr Ser Tyr Ala Asp Val Thr Phe 100 105 110 Leu Asp Asp GlnHis His Trp Gln Gln Leu Pro Gln Leu Ser Ala Ala 115 120 125 Ala Cys GlyCys Gly Gly 130 (2) INFORMATION FOR SEQ ID NO: 82: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 89 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: unknown (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 82: Cys Arg Leu Trp Ser Leu ThrLeu Pro Val Ala Glu Leu Gly Leu Gly 1 5 10 15 Tyr Ala Ser Glu Glu LysIle Ile Phe Arg Tyr Cys Ala Gly Ser Cys 20 25 30 Pro Gln Glu Val Arg ThrGln His Ser Leu Val Leu Ala Arg Leu Arg 35 40 45 Gly Gln Gly Arg Ala HisGly Arg Pro Cys Cys Gln Pro Thr Ser Tyr 50 55 60 Ala Asp Val Thr Phe LeuAsp Asp His His His Trp Gln Gln Leu Pro 65 70 75 80 Gln Leu Ser Ala AlaAla Cys Gly Cys 85 (2) INFORMATION FOR SEQ ID NO: 83: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 91 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: unknown (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 83: Cys Arg Leu Trp Ser Leu ThrLeu Pro Val Ala Glu Leu Gly Leu Gly 1 5 10 15 Tyr Ala Ser Glu Glu LysIle Ile Phe Arg Tyr Cys Ala Gly Ser Cys 20 25 30 Pro Gln Glu Val Arg ThrGln His Ser Leu Val Leu Ala Arg Leu Arg 35 40 45 Gly Gln Gly Arg Ala HisGly Arg Pro Cys Cys Gln Pro Thr Ser Tyr 50 55 60 Ala Asp Val Thr Phe LeuAsp Asp His His His Trp Gln Gln Leu Pro 65 70 75 80 Gln Leu Ser Ala AlaAla Cys Gly Cys Gly Gly 85 90 (2) INFORMATION FOR SEQ ID NO: 84: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 267 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 84: TGCCGACTGT GGAGCCTGACCCTACCAGTG GCTGAGCTGG GCCTGGGCTA TGCCTCGGAG 60 GAGAAGGTCA TCTTCCGATACTGTGCTGGC AGCTGTCCCC AAGAGGCCCG TACCCAGCAC 120 AGTCTGGTAC TGGCCCGGCTTCGAGGGCGG GGTCGAGCCC ATGGCCGACC CTGCTGCCAG 180 CCCACCAGCT ATGCTGATGTGACCTTCCTT GATGATCAGC ACCATTGGCA GCAGCTGCCT 240 CAGCTCTCAG CTGCAGCTTGTGGCTGT 267 (2) INFORMATION FOR SEQ ID NO: 85: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 267 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 85: TGCCGGCTGT GGAGCCTGAC CCTACCAGTGGCTGAGCTTG GCCTGGGCTA TGCCTCAGAG 60 GAGAAGATTA TCTTCCGATA CTGTGCTGGCAGCTGTCCCC AAGAGGTCCG TACCCAGCAC 120 AGTCTGGTGC TGGCCCGTCT TCGAGGGCAGGGTCGAGCTC ATGGCAGACC TTGCTGCCAG 180 CCCACCAGCT ATGCTGATGT GACCTTCCTTGATGACCACC ACCATTGGCA GCAGCTGCCT 240 CAGCTCTCAG CCGCAGCTTG TGGCTGT 267(2) INFORMATION FOR SEQ ID NO: 86: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 273 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 86: TGCCGGCTGT GGAGCCTGAC CCTACCAGTG GCTGAGCTTG GCCTGGGCTATGCCTCAGAG 60 GAGAAGATTA TCTTCCGATA CTGTGCTGGC AGCTGTCCCC AAGAGGTCCGTACCCAGCAC 120 AGTCTGGTGC TGGCCCGTCT TCGAGGGCAG GGTCGAGCTC ATGGCAGACCTTGCTGCCAG 180 CCCACCAGCT ATGCTGATGT GACCTTCCTT GATGACCACC ACCATTGGCAGCAGCTGCCT 240 CAGCTCTCAG CCGCAGCTTG TGGCTGTGGT GGC 273 (2) INFORMATIONFOR SEQ ID NO: 87: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 94 aminoacids (B) TYPE: amino acid (C) STRANDEDNESS: unknown (D) TOPOLOGY:linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:87: Cys Val Leu Thr Ala Ile His Leu Asn Val Thr Asp Leu Gly Leu Gly 1 510 15 Tyr Glu Thr Lys Glu Glu Leu Ile Phe Arg Tyr Cys Ser Gly Ser Cys 2025 30 Glu Ser Ala Glu Thr Met Tyr Asp Lys Ile Leu Lys Asn Leu Ser Arg 3540 45 Ser Arg Arg Leu Thr Ser Asp Lys Val Gly Gln Ala Cys Cys Arg Pro 5055 60 Val Ala Phe Asp Asp Asp Leu Ser Phe Leu Asp Asp Asn Leu Val Tyr 6570 75 80 His Ile Leu Arg Lys His Ser Ala Lys Arg Cys Gly Cys Ile 85 90(2) INFORMATION FOR SEQ ID NO: 88: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 95 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: unknown(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 88: Cys Gly Leu Arg Glu Leu Glu Val Arg Val SerGlu Leu Gly Leu Gly 1 5 10 15 Tyr Thr Ser Asp Glu Thr Val Leu Phe ArgTyr Cys Ala Gly Ala Cys 20 25 30 Glu Ala Ala Ile Arg Ile Tyr Asp Leu GlyLeu Arg Arg Leu Arg Gln 35 40 45 Arg Arg Arg Val Arg Arg Glu Arg Ala ArgAla His Pro Cys Cys Arg 50 55 60 Pro Thr Ala Tyr Glu Asp Glu Val Ser PheLeu Asp Val His Ser Arg 65 70 75 80 Tyr His Thr Leu Gln Glu Leu Ser AlaArg Glu Cys Ala Cys Val 85 90 95 (2) INFORMATION FOR SEQ ID NO: 89: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 91 amino acids (B) TYPE: aminoacid (C) STRANDEDNESS: unknown (D) TOPOLOGY: linear (ii) MOLECULE TYPE:protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 89: Cys Arg Leu Trp SerLeu Thr Leu Pro Val Ala Glu Leu Gly Leu Gly 1 5 10 15 Tyr Ala Ser GluGlu Lys Val Ile Phe Arg Tyr Cys Ala Gly Ser Cys 20 25 30 Pro Gln Glu AlaArg Thr Gln His Ser Leu Val Leu Ala Arg Leu Arg 35 40 45 Gly Arg Gly ArgAla His Gly Arg Pro Cys Cys Gln Pro Thr Ser Tyr 50 55 60 Ala Asp Val ThrPhe Leu Asp Asp Gln His His Trp Gln Gln Leu Pro 65 70 75 80 Gln Leu SerAla Ala Ala Cys Gly Cys Gly Gly 85 90 (2) INFORMATION FOR SEQ ID NO: 90:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 90: TGCCTCAGAGGAGAAGATTA TC 22 (2) INFORMATION FOR SEQ ID NO: 91: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 7 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: unknown (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 91: Ala Ser Glu Glu Lys Ile Ile 15 (2) INFORMATION FOR SEQ ID NO: 92: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 16 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: unknown(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 92: Leu Gly Leu Gly Tyr Glu Thr Lys Glu Glu LeuIle Phe Arg Tyr Cys 1 5 10 15 (2) INFORMATION FOR SEQ ID NO: 93: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 16 amino acids (B) TYPE: aminoacid (C) STRANDEDNESS: unknown (D) TOPOLOGY: linear (ii) MOLECULE TYPE:protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 93: Leu Gly Leu Gly TyrThr Ser Asp Glu Thr Val Leu Phe Arg Tyr Cys 1 5 10 15 (2) INFORMATIONFOR SEQ ID NO: 94: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 16 aminoacids (B) TYPE: amino acid (C) STRANDEDNESS: unknown (D) TOPOLOGY:linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:94: Leu Gly Leu Gly Tyr Ala Ser Glu Glu Lys Ile Ile Phe Arg Tyr Cys 1 510 15 (2) INFORMATION FOR SEQ ID NO: 95: (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 23 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 95: AGTCGGGGTT GGGGTATGCC TCA 23 (2) INFORMATIONFOR SEQ ID NO: 96: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 26 basepairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:96: TATGCCTCAG AGGAGAAGAT TATCTT 26 (2) INFORMATION FOR SEQ ID NO: 97:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 336 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 97: CCTCAGAGGAGAAGATTATC TTCCGATACT GTGCTGGCAG CTGTCCCCAA GAGGTCCGTA 60 CCCAGCACAGTCTGGTGCTG GCCCGTCTTC GAGGGCAGGG TCGAGCTCAT GGCAGACCTT 120 GCTGCCAGCCCACCAGCTAT GCTGATGTGA CCTTCCTTGA TGACCACCAC CATTGGCAGC 180 AGCTGCCTCAGCTCTCAGCC GCAGCTTGTG GCTGTGGTGG CTGAAGGCGG CCAGCCTGGT 240 CTCTCAGAATCACAAGCAAG AGGCAGCCTT TGAAAGGCTC AGGTGACGTT ATTAGAAACT 300 TGCATAGGAGAAGATTAAGA AGAGAAAGGG GACCTG 336 (2) INFORMATION FOR SEQ ID NO: 98: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 17 amino acids (B) TYPE: aminoacid (C) STRANDEDNESS: unknown (D) TOPOLOGY: linear (ii) MOLECULE TYPE:protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 98: Ala Cys Cys Arg ProVal Ala Phe Asp Asp Asp Leu Ser Phe Leu Asp 1 5 10 15 Asp (2)INFORMATION FOR SEQ ID NO: 99: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:17 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: unknown (D)TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 99: Pro Cys Cys Arg Pro Thr Ala Tyr Glu Asp Glu Val Ser PheLys Asp 1 5 10 15 Val (2) INFORMATION FOR SEQ ID NO: 100: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 16 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: unknown (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 100: Pro Cys Cys Gln Pro Thr SerTyr Ala Asp Val Thr Phe Leu Asp Asp 1 5 10 15 (2) INFORMATION FOR SEQ IDNO: 101: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 26 base pairs (B)TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 101:TCATCAAGGA AGGTCACATC AGCATA 26 (2) INFORMATION FOR SEQ ID NO: 102: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 32 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 102: CCACCACAGC CACAAGCTGCGGSTGAGAGC TG 32 (2) INFORMATION FOR SEQ ID NO: 103: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 5 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: unknown (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 103: Ala Leu Ala Gly Ser 1 5 (2)INFORMATION FOR SEQ ID NO: 104: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 43 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: unknown(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 104: Val Arg Ile Pro Gly Gly Leu Pro Thr Pro GlnPhe Leu Leu Ser Lys 1 5 10 15 Pro Ser Leu Cys Leu Thr Ile Leu Leu TyrLeu Ala Leu Gly Asn Asn 20 25 30 His Val Arg Leu Pro Arg Ala Leu Ala GlySer 35 40 (2) INFORMATION FOR SEQ ID NO: 105: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 544 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 105: GAGGGACCTG GACGCCCCAT CAGGGTAAGAATTCCTGGGG GCCTCCCGAC TCCCCAATTC 60 CTTCTCTCAA AGCCCTCACT TTGCCTTACAATCCTACTCT ACCTTGCACT AGGTAACAAC 120 CATGTCCGTC TTCCAAGAGC CTTGGCTGGTTCATGCCGAC TGTGGAGCCT GACCCTACCA 180 GTGGCTGAGC TGGGCCTGGG CTATGCCTCGGAGGAGAAGG TCATCTTCCG ATACTGTGCT 240 GGCAGCTGTC CCCAAGAGGC CCGTACCCAGCACAGTCTGG TACTGGCCCG GCTTCGAGGG 300 CGGGGTCGAG CCCATGGCCG ACCCTGCTGCCAGCCCACCA GCTATGCTGA TGTGACCTTC 360 CTTGATGATC AGCACCATTG GCAGCAGCTGCCTCAGCTCT CAGCTGCAGC TTGTGGCTGT 420 GGTGGCTGAA GGAGGCCAGT CTGGTGTCTCAGAATCACAA GCATGAGACA GGCTGGGCTT 480 TGAAAGGCTC AGGTGACATT ACTAGAAATTTGCATAGGTA AAGATAAGAA GGGAAAGGAC 540 CAGG 544 (2) INFORMATION FOR SEQ IDNO: 106: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 336 base pairs (B)TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 106:CCTCAGAGGA GAAGATTATC TTCCGATACT GTGCTGGCAG CTGTCCCCAA GAGGTCCGTA 60CCCAGCACAG TCTGGTGCTG GCCCGTCTTC GAGGGCAGGG TCGAGCTCAT GGCAGACCTT 120GCTGCCAGCC CACCAGCTAT GCTGATGTGA CCTTCCTTGA TGACCACCAC CATTGGCAGC 180AGCTGCCTCA GCTCTCAGCC GCAGCTTGTG GCTGTGGTGG CTGAAGGCGG CCAGCCTGGT 240CTCTCAGAAT CACAAGCAAG AGGCAGCCTT TGAAAGGCTC AGGTGACGTT ATTAGAAACT 300TGCATAGGAG AAGATTAAGA AGAGAAAGGG GACCTG 336 (2) INFORMATION FOR SEQ IDNO: 107: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 391 base pairs (B)TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 107:TGCCGGCTGT GGAGCCTGAC CCTACCAGTG GCTGAGCTTG GCCTGGGCTA TGCCTCAGAG 60GAGAAGATTA TCTTCCGATA CTGTGCTGGC AGCTGTCCCC AAGAGGTCCG TACCCAGCAC 120AGTCTGGTGC TGGCCCGTCT TCGAGGGCAG GGTCGAGCTC ATGGCAGACC TTGCTGCCAG 180CCCACCAGCT ATGCTGATGT GACCTTCCTT GATGACCACC ACCATTGGCA GCAGCTGCCT 240CAGCTCTCAG CCGCAGCTTG TGGCTGTGGT GGCTGAAGGC GGCCAGCCTG GTCTCTCAGA 300ATCACAAGCA AGAGGCAGCC TTTGAAAGGC TCAGGTGACG TTATTAGAAA CTTGCATAGG 360AGAAGATTAA GAAGAGAAAG GGGACCTGAT T 391 (2) INFORMATION FOR SEQ ID NO:108: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE:amino acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: linear (ii) MOLECULETYPE: protein (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 2(D) OTHER INFORMATION: /note= “SERINE, THREONINE, OR ALANINE” (ix)FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 3 (D) OTHERINFORMATION: /note= “GLUTAMIC ACID OR ASPARTIC ACID” (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 108: Val Xaa Xaa Leu Gly Leu Gly Tyr 1 5 (2)INFORMATION FOR SEQ ID NO: 109: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 8 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: unknown (D)TOPOLOGY: linear (ii) MOLECULE TYPE: protein (ix) FEATURE: (A) NAME/KEY:Modified-site (B) LOCATION: 5 (D) OTHER INFORMATION: /note= “ALANINE ORSERINE” (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 7 (D)OTHER INFORMATION: /note= “ALANINE OR SERINE” (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 109: Phe Arg Tyr Cys Xaa Gly Xaa Cys 1 5 (2) INFORMATION FORSEQ ID NO: 110: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids(B) TYPE: amino acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: linear (ii)MOLECULE TYPE: protein (ix) FEATURE: (A) NAME/KEY: Modified-site (B)LOCATION: 2 (D) OTHER INFORMATION: /note= “ASPARTIC ACID, GLUTAMIC ACIDOR NO AMINO ACID” (ix) FEATURE: (A) NAME/KEY: Modified-site (B)LOCATION: 3 (D) OTHER INFORMATION: /note= “VALINE OR LEUCINE” (ix)FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 4 (D) OTHERINFORMATION: /note= “SERINE OR THREONINE” (ix) FEATURE: (A) NAME/KEY:Modified-site (B) LOCATION: 8 (D) OTHER INFORMATION: /note= “valine oraspartic acid” (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 110: Asp Xaa XaaXaa Phe Leu Asp Xaa 1 5 (2) INFORMATION FOR SEQ ID NO: 111: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 142 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: unknown (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 111: Glu Gly Pro Gly Arg Pro IleArg Val Arg Ile Pro Gly Gly Leu Pro 1 5 10 15 Thr Pro Gln Phe Leu LeuSer Lys Pro Ser Leu Cys Leu Thr Ile Leu 20 25 30 Leu Tyr Leu Ala Leu GlyAsn Asn His Val Arg Leu Pro Arg Ala Leu 35 40 45 Ala Gly Ser Cys Arg LeuTrp Ser Leu Thr Leu Pro Val Ala Glu Leu 50 55 60 Gly Leu Gly Tyr Ala SerGlu Glu Lys Val Ile Phe Arg Tyr Cys Ala 65 70 75 80 Gly Ser Cys Pro GlnGlu Ala Arg Thr Gln His Ser Leu Val Leu Ala 85 90 95 Arg Leu Arg Gly ArgGly Arg Ala His Gly Arg Pro Cys Cys Gln Pro 100 105 110 Thr Ser Tyr AlaAsp Val Thr Phe Leu Asp Asp Gln His His Trp Gln 115 120 125 Gln Leu ProGln Leu Ser Ala Ala Ala Cys Gly Cys Gly Gly 130 135 140 (2) INFORMATIONFOR SEQ ID NO: 112: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 5 aminoacids (B) TYPE: amino acid (C) STRANDEDNESS: unknown (D) TOPOLOGY:linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:112: Ala Leu Pro Gly Leu 1 5 (2) INFORMATION FOR SEQ ID NO: 113: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 12 amino acids (B) TYPE: aminoacid (C) STRANDEDNESS: unknown (D) TOPOLOGY: linear (ii) MOLECULE TYPE:protein (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 2 (D)OTHER INFORMATION: /note= “THREONINE, GLUTAMIC ACID OR LYSINE” (ix)FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 3 (D) OTHERINFORMATION: /note= “VALINE, LEUCINE OR ISOLEUCINE” (ix) FEATURE: (A)NAME/KEY: Modified-site (B) LOCATION: 4 (D) OTHER INFORMATION: /note=“LEUCINE OR ISOLEUCINE” (ix) FEATURE: (A) NAME/KEY: Modified-site (B)LOCATION: 9 (D) OTHER INFORMATION: /note= “ALANINE OR SERINE” (ix)FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 11 (D) OTHERINFORMATION: /note= “ALANINE OR SERINE” (xi) SEQUENCE DESCRIPTION: SEQID NO: 113: Glu Xaa Xaa Xaa Phe Arg Tyr Cys Xaa Gly Xaa Cys 1 5 10 (2)INFORMATION FOR SEQ ID NO: 114: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 16 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: unknown(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (ix) FEATURE: (A)NAME/KEY: Modified-site (B) LOCATION: 3 (D) OTHER INFORMATION: /note=“ARGININE OR GLUTAMINE” (ix) FEATURE: (A) NAME/KEY: Modified-site (B)LOCATION: 5 (D) OTHER INFORMATION: /note= “THREONINE, VALINE ORISOLEUCINE” (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 6(D) OTHER INFORMATION: /note= “ALANINE OR SERINE” (ix) FEATURE: (A)NAME/KEY: Modified-site (B) LOCATION: 7 (D) OTHER INFORMATION: /note=“TYROSINE OR PHENYLALANINE” (ix) FEATURE: (A) NAME/KEY: Modified-site(B) LOCATION: 8 (D) OTHER INFORMATION: /note= “GLUTAMIC ACID, ASPARTICACID OR ALANINE” (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION:10 (D) OTHER INFORMATION: /note= “GLUTAMIC ACID, ASPARTIC ACID OR NOAMINO ACID” (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 11(D) OTHER INFORMATION: /note= “VALINE OR LEUCINE” (ix) FEATURE: (A)NAME/KEY: Modified-site (B) LOCATION: 12 (D) OTHER INFORMATION: /note=“SERINE OR THREONINE” (ix) FEATURE: (A) NAME/KEY: Modified-site (B)LOCATION: 16 (D) OTHER INFORMATION: /note= “ASPARTIC ACID OR VALINE”(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 114: Cys Cys Xaa Pro Xaa Xaa XaaXaa Asp Xaa Xaa Xaa Phe Leu Asp Xaa 1 5 10 15 (2) INFORMATION FOR SEQ IDNO: 115: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 base pairs (B)TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 115:GTNDGNGANY TGGGNYTGGG NTA 23 (2) INFORMATION FOR SEQ ID NO: 116: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 116: GANBTNWCNT TYYTNGANG 19(2) INFORMATION FOR SEQ ID NO: 117: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 117: GANBTNWCNT TYYTNGANGW 20 (2) INFORMATION FOR SEQ ID NO:118: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 118: TTYMGNTAYTGYDSNGGNDS NTG 23 (2) INFORMATION FOR SEQ ID NO: 119: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 119: GTNDGNGANY TGGGNYTNGG 20 (2)INFORMATION FOR SEQ ID NO: 120: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 23 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 120: GTNDGNGANY TGGGNYTGGG NTT 23 (2) INFORMATION FOR SEQ IDNO: 121: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B)TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 121:WCNTCNARRA ANGWNAVNTC 20 (2) INFORMATION FOR SEQ ID NO: 122: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 122: WCNTCNARRA ANGWNAVNT 19(2) INFORMATION FOR SEQ ID NO: 123: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 23 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 123: CANSHNCCNS HRCARTANCK RAA 23 (2) INFORMATION FOR SEQ IDNO: 124: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 25 base pairs (B)TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 124:CANSHNCCNS HRCARTANCK RAANA 25 (2) INFORMATION FOR SEQ ID NO: 125: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid(C) STRANDEDNESS: unknown (D) TOPOLOGY: linear (ii) MOLECULE TYPE:protein (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 2 (D)OTHER INFORMATION: /note= “THREONINE, SERINE OR ALANINE” (ix) FEATURE:(A) NAME/KEY: Modified-site (B) LOCATION: 3 (D) OTHER INFORMATION:/note= “GLUTAMIC ACID OR ASPARTIC ACID” (xi) SEQUENCE DESCRIPTION: SEQID NO: 125: Val Xaa Xaa Leu Gly Leu Gly Tyr 1 5 (2) INFORMATION FOR SEQID NO: 126: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 7 amino acids (B)TYPE: amino acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: linear (ii)MOLECULE TYPE: protein (ix) FEATURE: (A) NAME/KEY: Modified-site (B)LOCATION: 1 (D) OTHER INFORMATION: /note= “ASPARTIC ACID OR GLUTAMICACID” (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 2 (D)OTHER INFORMATION: /note= “VALINE OR LEUCINE” (ix) FEATURE: (A)NAME/KEY: Modified-site (B) LOCATION: 3 (D) OTHER INFORMATION: /note=“THREONINE OR SERINE” (ix) FEATURE: (A) NAME/KEY: Modified-site (B)LOCATION: 6 (D) OTHER INFORMATION: /note= “ASPARTIC ACID OR GLUTAMICACID” (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 7 (D)OTHER INFORMATION: /note= “ASPARTIC ACID OR VALINE” (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 126: Xaa Xaa Xaa Phe Leu Xaa Xaa 1 5 (2)INFORMATION FOR SEQ ID NO: 127: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 8 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: unknown (D)TOPOLOGY: linear (ii) MOLECULE TYPE: protein (ix) FEATURE: (A) NAME/KEY:Modified-site (B) LOCATION: 5 (D) OTHER INFORMATION: /note= “SERINE ORALANINE” (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 7 (D)OTHER INFORMATION: /note= “SERINE OR ALANINE” (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 127: Phe Arg Tyr Cys Xaa Gly Xaa Cys 1 5 (2) INFORMATION FORSEQ ID NO: 128: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 7 amino acids(B) TYPE: amino acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: linear (ii)MOLECULE TYPE: protein (ix) FEATURE: (A) NAME/KEY: Modified-site (B)LOCATION: 2 (D) OTHER INFORMATION: /note= “THREONINE, SERINE OR ALANINE”(ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 3 (D) OTHERINFORMATION: /note= “ASPARTIC ACID OR GLUTAMIC ACID” (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 128: Val Xaa Xaa Leu Gly Leu Gly 1 5 (2)INFORMATION FOR SEQ ID NO: 129: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 8 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: unknown (D)TOPOLOGY: linear (ii) MOLECULE TYPE: protein (ix) FEATURE: (A) NAME/KEY:Modified-site (B) LOCATION: 2 (D) OTHER INFORMATION: /note= “THREONINE,SERINE OR ALANINE” (ix) FEATURE: (A) NAME/KEY: Modified-site (B)LOCATION: 3 (D) OTHER INFORMATION: /note= “GLUTAMIC ACID OR ASPARTICACID” (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 129: Val Xaa Xaa Leu Gly LeuGly Phe 1 5 (2) INFORMATION FOR SEQ ID NO: 130: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 9 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: unknown (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein(ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 1 (D) OTHERINFORMATION: /note= “ISOLEUCINE OR LEUCINE” (ix) FEATURE: (A) NAME/KEY:Modified-site (B) LOCATION: 6 (D) OTHER INFORMATION: /note= “SERINE ORALANINE” (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 8 (D)OTHER INFORMATION: /note= “SERINE OR ALANINE” (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 130: Xaa Phe Arg Tyr Cys Xaa Gly Xaa Cys 1 5 (2) INFORMATIONFOR SEQ ID NO: 131: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 559 basepairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQID NO: 131: ATGGCTGCAG GAAGACTTCG GATCCTGTGT CTGCTGCTCC TGTCCTTGCACCCGAGCCTC 60 GGCTGGGTCC TTGATCTTCA AGAGGCTTCT GTGGCAGATA AGCTCTCATTTGGGAAGATG 120 GCAGAGACTA GAGGGACCTG GACGCCCCAT CAGGGTAAGA ATTCCTGGGGGCCTCCCGAC 180 TCCCCAATTC CTTCTCTCAA AGCCCTCACT TTGCCTTACA ATCCTACTCTACCTTGCACT 240 AGGTAACAAC CATGTCCGTC TTCCAAGAGC CTTGGCTGGT TCATGCCGACTGTGGAGCCT 300 GACCCTACCA GTGGCTGAGC TGGGCCTGGG CTATGCCTCG GAGGAGAAGGTCATCTTCCG 360 ATACTGTGCT GGCAGCTGTC CCCAAGAGGC CCGTACCCAG CACAGTCTGGTACTGGCCCG 420 GCTTCGAGGG CGGGGTCGAG CCCATGGCCG ACCCTGCTGC CAGCCCACCAGCTATGCTGA 480 TGTGACCTTC CTTGATGATC AGCACCATTG GCAGCAGCTG CCTCAGCTCTCAGCTGCAGC 540 TTGTGGCTGT GGTGGCTGA 559 (2) INFORMATION FOR SEQ ID NO:132: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 81 amino acids (B) TYPE:amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 132: Met Ala Ala GlyArg Leu Arg Ile Leu Cys Leu Leu Leu Leu Ser Leu 1 5 10 15 His Pro SerLeu Gly Trp Val Leu Asp Leu Gln Glu Ala Ser Val Ala 20 25 30 Asp Lys LeuSer Phe Gly Lys Met Ala Glu Thr Arg Gly Thr Trp Thr 35 40 45 Pro His GlnGly Lys Asn Ser Trp Gly Pro Pro Asp Ser Pro Ile Pro 50 55 60 Ser Leu LysAla Leu Thr Leu Pro Tyr Asn Pro Thr Leu Pro Cys Thr 65 70 75 80 Arg (2)INFORMATION FOR SEQ ID NO: 133: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 185 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 133: Trp Leu Gln Glu Asp Phe Gly Ser Cys Val CysCys Ser Cys Pro Cys 1 5 10 15 Thr Arg Ala Ser Ala Gly Ser Leu Ile PheLys Arg Leu Leu Trp Gln 20 25 30 Ile Ser Ser His Leu Gly Arg Trp Gln ArgLeu Glu Gly Pro Gly Arg 35 40 45 Pro Ile Arg Val Arg Ile Pro Gly Gly LeuPro Thr Pro Gln Phe Leu 50 55 60 Leu Ser Lys Pro Ser Leu Cys Leu Thr IleLeu Leu Tyr Leu Ala Leu 65 70 75 80 Gly Asn Asn His Val Arg Leu Pro ArgAla Leu Ala Gly Ser Cys Arg 85 90 95 Leu Trp Ser Leu Thr Leu Pro Val AlaGlu Leu Gly Leu Gly Tyr Ala 100 105 110 Ser Glu Glu Lys Val Ile Phe ArgTyr Cys Ala Gly Ser Cys Pro Gln 115 120 125 Glu Ala Arg Thr Gln His SerLeu Val Leu Ala Arg Leu Arg Gly Arg 130 135 140 Gly Arg Ala His Gly ArgPro Cys Cys Gln Pro Thr Ser Tyr Ala Asp 145 150 155 160 Val Thr Phe LeuAsp Asp Gln His His Trp Gln Gln Leu Pro Gln Leu 165 170 175 Ser Ala AlaAla Cys Gly Cys Gly Gly 180 185 (2) INFORMATION FOR SEQ ID NO: 134: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 559 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 134: ATGGCTGCAGGAAGACTTCG GATCTTGTTT CTGCTGCTCC TGTCCTTGCA CCTGGGCCTT 60 GGCTGGGTCCTTGATCTTCA AGAGGCTCCT GCGGCAGATG AGCTCTCATC TGGGAAAATG 120 GCAGAGACTGGAAGGACCTG GAAGCCCCAT CAGGGTAAGA ATTCTTGGGG GCCTCCTAAC 180 TCTACAGTTCTTCCTCTCAA AGCCCTCACT TTGCCTCACA ATCCTATTCT ACCTTGCACT 240 AGGTAACAACAATGTCCGCC TTCCAAGAGC CTTACCTGGT TTGTGCCGGC TGTGGAGCCT 300 GACCCTACCAGTGGCTGAGC TTGGCCTGGG CTATGCCTCA GAGGAGAAGA TTATCTTCCG 360 ATACTGTGCTGGCAGCTGTC CCCAAGAGGT CCGTACCCAG CACAGTCTGG TGCTGGCCCG 420 TCTTCGAGGGCAGGGTCGAG CTCATGGCAG ACCTTGCTGC CAGCCCACCA GCTATGCTGA 480 TGTGACCTTCCTTGATGACC ACCACCATTG GCAGCAGCTG CCTCAGCTCT CAGCCGCAGC 540 TTGTGGCTGTGGTGGCTGA 559 (2) INFORMATION FOR SEQ ID NO: 135: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 81 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 135: Met Ala Ala Gly Arg Leu ArgIle Leu Phe Leu Leu Leu Leu Ser Leu 1 5 10 15 His Leu Gly Leu Gly TrpVal Leu Asp Leu Gln Glu Ala Pro Ala Ala 20 25 30 Asp Glu Leu Ser Ser GlyLys Met Ala Glu Thr Gly Arg Thr Trp Lys 35 40 45 Pro His Gln Gly Lys AsnSer Trp Gly Pro Pro Asn Ser Thr Val Leu 50 55 60 Pro Leu Lys Ala Leu ThrLeu Pro His Asn Pro Ile Leu Pro Cys Thr 65 70 75 80 Arg (2) INFORMATIONFOR SEQ ID NO: 136: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 185 aminoacids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 136:Trp Leu Gln Glu Asp Phe Gly Ser Cys Phe Cys Cys Ser Cys Pro Cys 1 5 1015 Thr Trp Ala Leu Ala Gly Ser Leu Ile Phe Lys Arg Leu Leu Arg Gln 20 2530 Met Ser Ser His Leu Gly Lys Trp Gln Arg Leu Glu Gly Pro Gly Ser 35 4045 Pro Ile Arg Val Arg Ile Leu Gly Gly Leu Leu Thr Leu Gln Phe Phe 50 5560 Leu Ser Lys Pro Ser Leu Cys Leu Thr Ile Leu Phe Tyr Leu Ala Leu 65 7075 80 Gly Asn Asn Asn Val Arg Leu Pro Arg Ala Leu Pro Gly Leu Cys Arg 8590 95 Leu Trp Ser Leu Thr Leu Pro Val Ala Glu Leu Gly Leu Gly Tyr Ala100 105 110 Ser Glu Glu Lys Ile Ile Phe Arg Tyr Cys Ala Gly Ser Cys ProGln 115 120 125 Glu Val Arg Thr Gln His Ser Leu Val Leu Ala Arg Leu ArgGly Gln 130 135 140 Gly Arg Ala His Gly Arg Pro Cys Cys Gln Pro Thr SerTyr Ala Asp 145 150 155 160 Val Thr Phe Leu Asp Asp His His His Trp GlnGln Leu Pro Gln Leu 165 170 175 Ser Ala Ala Ala Cys Gly Cys Gly Gly 180185 (2) INFORMATION FOR SEQ ID NO: 137: (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 23 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 137: AATCCCCAGG ACAGGCAGGG AAT 23 (2)INFORMATION FOR SEQ ID NO: 138: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 35 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 138: CGGTACCCAG ATCTTCAGCC ACCACAGCCACAAGC 35 (2) INFORMATION FOR SEQ ID NO: 139: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 76 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: othernucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 139: GGACTATCATATGGCCCACC ACCACCACCA CCACCACCAC GACGACGACG ACAAGGCCTT 60 GGCTGGTTCATGCCGA 76 2) INFORMATION FOR SEQ ID NO: 140: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 35 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: othernucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 140: CGGTACCCAGATCTTCAGCC ACCACAGCCA CAAGC 35 (2) INFORMATION FOR SEQ ID NO: 141: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 96 amino acids (B) TYPE: aminoacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 141: Ala Leu Ala Gly SerCys Arg Leu Trp Ser Leu Thr Leu Pro Val Ala 1 5 10 15 Glu Leu Gly LeuGly Tyr Ala Ser Glu Glu Lys Val Ile Phe Arg Tyr 20 25 30 Cys Ala Gly SerCys Pro Gln Glu Ala Arg Thr Gln His Ser Leu Val 35 40 45 Leu Ala Arg LeuArg Gly Arg Gly Arg Ala His Gly Arg Pro Cys Cys 50 55 60 Arg Pro Thr AlaTyr Glu Asp Glu Val Ser Phe Leu Asp Val His Ser 65 70 75 80 Arg Tyr HisThr Leu Gln Glu Leu Ser Ala Arg Glu Cys Ala Cys Val 85 90 95 (2)INFORMATION FOR SEQ ID NO: 142: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 23 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 142: TAATACGACT CACTATAGGG GAA 23 (2)INFORMATION FOR SEQ ID NO: 143: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 49 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 143: TCGTCTTCGT AAGCAGTCGG ACGGCAGCAGGGTCGGCCAT GGGCTCGAC 49 (2) INFORMATION FOR SEQ ID NO: 144: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 29 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: othernucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 144: TGCTGCCGTCCGACTGCTTA CGAAGACGA 29 (2) INFORMATION FOR SEQ ID NO: 145: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: othernucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 145: GTTATGCTAGTTATTGCTCA GCGGT 25 (2) INFORMATION FOR SEQ ID NO: 146: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 100 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 146: Pro Gly Ala Arg Pro Cys GlyLeu Arg Glu Leu Glu Val Arg Val Ser 1 5 10 15 Glu Leu Gly Leu Gly TyrThr Ser Asp Glu Thr Val Leu Phe Arg Tyr 20 25 30 Cys Ala Gly Ala Cys GluAla Ala Ile Arg Ile Tyr Asp Leu Gly Leu 35 40 45 Arg Arg Leu Arg Gln ArgArg Arg Val Arg Arg Glu Arg Ala Arg Ala 50 55 60 His Pro Cys Cys Gln ProThr Ser Tyr Ala Asp Val Thr Phe Leu Asp 65 70 75 80 Asp Gln His His TrpGln Gln Leu Pro Gln Leu Ser Ala Ala Ala Cys 85 90 95 Gly Cys Gly Gly 100(2) INFORMATION FOR SEQ ID NO: 147: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 50 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 147: CACATCAGCA TAGCTGGTGG GCTGGCAGCACGGGTGAGCA CGAGCACGTT 50 (2) INFORMATION FOR SEQ ID NO: 148: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 25 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:other nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 148: TGCTGCCAGCCCACCAGCTA TGCTG 25 (2) INFORMATION FOR SEQ ID NO: 149: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 23 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: othernucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 149: CCTCGGAGGAGAAGGTCATC TTC 23 (2) INFORMATION FOR SEQ ID NO: 150: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 98 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 150: Cys Cys Val Arg Gln Leu TyrIle Asp Phe Arg Lys Asp Leu Gly Trp 1 5 10 15 Lys Trp Ile His Glu ProLys Gly Tyr His Ala Asn Phe Cys Leu Gly 20 25 30 Pro Cys Pro Tyr Ile TrpSer Leu Asp Thr Gln Tyr Ser Lys Val Leu 35 40 45 Ala Leu Tyr Asn Gln HisAsn Pro Gly Ala Ser Ala Ala Pro Cys Cys 50 55 60 Val Pro Gln Ala Leu GluPro Leu Pro Ile Val Tyr Tyr Val Gly Arg 65 70 75 80 Lys Pro Lys Val GluGln Leu Ser Asn Met Ile Val Arg Ser Cys Lys 85 90 95 Cys Ser (2)INFORMATION FOR SEQ ID NO: 151: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 98 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 151: Cys Cys Leu Arg Pro Leu Tyr Ile Asp Phe Lys Arg Asp LeuGly Trp 1 5 10 15 Lys Trp Ile His Glu Pro Lys Gly Tyr Asn Ala Asn PheCys Ala Gly 20 25 30 Ala Cys Pro Tyr Leu Trp Ser Ser Asp Thr Gln His SerArg Val Leu 35 40 45 Ser Leu Tyr Asn Thr Ile Asn Pro Glu Ala Ser Ala SerPro Cys Cys 50 55 60 Val Ser Gln Asp Leu Glu Pro Leu Thr Ile Leu Tyr TyrIle Gly Lys 65 70 75 80 Thr Pro Lys Ile Glu Gln Leu Ser Asn Met Ile ValLys Ser Cys Lys 85 90 95 Cys Ser (2) INFORMATION FOR SEQ ID NO: 152: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 98 amino acids (B) TYPE: aminoacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 152: Cys Cys Val Arg ProLeu Tyr Ile Asp Phe Arg Gln Asp Leu Gly Trp 1 5 10 15 Lys Trp Val HisGlu Pro Lys Gly Tyr Tyr Ala Asn Phe Cys Ser Gly 20 25 30 Pro Cys Pro TyrLeu Arg Ser Ala Asp Thr Thr His Ser Thr Val Leu 35 40 45 Gly Leu Tyr AsnThr Leu Asn Pro Glu Ala Ser Ala Ser Pro Cys Cys 50 55 60 Val Pro Gln AspLeu Glu Pro Leu Thr Ile Leu Tyr Tyr Val Gly Arg 65 70 75 80 Thr Pro LysVal Glu Gln Leu Ser Asn Met Val Val Lys Ser Cys Lys 85 90 95 Cys Ser (2)INFORMATION FOR SEQ ID NO: 153: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 106 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 153: Cys Cys Lys Lys Gln Phe Phe Val Ser Phe LysAsp Ile Gly Trp Asn 1 5 10 15 Asp Trp Ile Ile Ala Pro Ser Gly Tyr HisAla Asn Tyr Cys Glu Gly 20 25 30 Glu Cys Pro Ser His Ile Ala Gly Thr SerGly Ser Ser Leu Ser Phe 35 40 45 His Ser Thr Val Ile Asn His Tyr Arg MetArg Gly His Ser Pro Phe 50 55 60 Ala Asn Leu Lys Ser Cys Cys Val Pro ThrLys Leu Arg Pro Met Ser 65 70 75 80 Met Leu Tyr Tyr Asp Asp Gly Gln AsnIle Ile Lys Lys Asp Ile Gln 85 90 95 Asn Met Ile Val Glu Glu Cys Gly CysSer 100 105 (2) INFORMATION FOR SEQ ID NO: 154: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 105 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 154: Cys Cys Arg Gln Gln Phe PheIle Asp Phe Arg Leu Ile Gly Trp Asn 1 5 10 15 Asp Trp Ile Ile Ala ProThr Gly Tyr Tyr Gly Asn Tyr Cys Glu Gly 20 25 30 Ser Cys Pro Ala Tyr LeuAla Gly Val Pro Gly Ser Ala Ser Ser Phe 35 40 45 His Thr Ala Val Val AsnGln Tyr Arg Met Arg Gly Leu Asn Pro Gly 50 55 60 Thr Val Asn Ser Cys CysIle Pro Thr Lys Leu Ser Thr Met Ser Met 65 70 75 80 Leu Tyr Phe Asp AspGlu Tyr Asn Ile Val Lys Arg Asp Val Pro Asn 85 90 95 Met Ile Val Glu GluCys Gly Cys Ala 100 105 (2) INFORMATION FOR SEQ ID NO: 155: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 101 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 155: Cys Arg Arg Val Lys Phe GlnVal Asp Phe Asn Leu Ile Gly Trp Gly 1 5 10 15 Ser Trp Ile Ile Tyr ProLys Gln Tyr Asn Ala Tyr Arg Cys Glu Gly 20 25 30 Glu Cys Pro Asn Pro ValGly Glu Glu Phe His Pro Thr Asn His Ala 35 40 45 Tyr Ile Gln Ser Leu LeuLys Arg Tyr Gln Pro His Arg Val Pro Ser 50 55 60 Thr Cys Cys Ala Pro ValLys Thr Lys Pro Leu Ser Met Leu Tyr Val 65 70 75 80 Asp Asn Gly Arg ValLeu Leu Glu His His Lys Asp Met Ile Val Glu 85 90 95 Glu Cys Gly Cys Leu100 (2) INFORMATION FOR SEQ ID NO: 156: (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 101 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 156: Cys Lys Arg His Pro Leu Tyr Val Asp Phe SerAsp Val Gly Trp Asn 1 5 10 15 Asp Trp Ile Val Ala Pro Pro Gly Tyr HisAla Phe Tyr Cys His Gly 20 25 30 Glu Cys Pro Phe Pro Leu Ala Asp His LeuAsn Ser Thr Asn His Ala 35 40 45 Ile Val Gln Thr Leu Val Asn Ser Val AsnSer Lys Ile Pro Lys Ala 50 55 60 Cys Cys Val Pro Thr Glu Leu Ser Ala IleSer Met Leu Tyr Leu Asp 65 70 75 80 Glu Asn Glu Lys Val Val Leu Lys AsnTyr Gln Asp Met Val Val Glu 85 90 95 Gly Cys Gly Cys Arg 100 (2)INFORMATION FOR SEQ ID NO: 157: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 101 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 157: Cys Arg Arg His Ser Leu Tyr Val Asp Phe SerAsp Val Gly Trp Asn 1 5 10 15 Asp Trp Ile Val Ala Pro Pro Gly Tyr GlnAla Phe Tyr Cys His Gly 20 25 30 Asp Cys Pro Phe Pro Leu Ala Asp His LeuAsn Ser Thr Asn His Ala 35 40 45 Ile Val Gln Thr Leu Val Asn Ser Val AsnSer Ser Ile Pro Lys Ala 50 55 60 Cys Cys Val Pro Thr Glu Leu Ser Ala IleSer Met Leu Tyr Leu Asp 65 70 75 80 Glu Tyr Asp Lys Val Val Leu Lys AsnTyr Gln Glu Met Val Val Glu 85 90 95 Gly Cys Gly Cys Arg 100 (2)INFORMATION FOR SEQ ID NO: 158: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 102 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 158: Cys Arg Arg His Ser Leu Tyr Val Asp Phe SerAsp Val Gly Trp Asp 1 5 10 15 Asp Trp Ile Val Ala Pro Leu Gly Tyr AspAla Tyr Tyr Cys His Gly 20 25 30 Lys Cys Pro Phe Pro Leu Ala Asp His PheAsn Ser Thr Asn His Ala 35 40 45 Val Val Gln Thr Leu Val Asn Asn Met AsnPro Gly Lys Val Pro Lys 50 55 60 Ala Cys Cys Val Pro Thr Gln Leu Asp SerVal Ala Met Leu Tyr Leu 65 70 75 80 Asn Asp Gln Ser Thr Val Val Leu LysAsn Tyr Gln Glu Met Thr Val 85 90 95 Val Gly Cys Gly Cys Arg 100 (2)INFORMATION FOR SEQ ID NO: 159: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 102 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 159: Cys Lys Lys His Glu Leu Tyr Val Ser Phe ArgAsp Leu Gly Trp Gln 1 5 10 15 Asp Trp Ile Ile Ala Pro Glu Gly Tyr AlaAla Phe Tyr Cys Asp Gly 20 25 30 Glu Cys Ser Phe Pro Leu Asn Ala His MetAsn Ala Thr Asn His Ala 35 40 45 Ile Val Gln Thr Leu Val His Leu Met PhePro Asp His Val Pro Lys 50 55 60 Pro Cys Cys Ala Pro Thr Lys Leu Asn AlaIle Ser Val Leu Tyr Phe 65 70 75 80 Asp Asp Ser Ser Asn Val Ile Leu LysLys Tyr Arg Asn Met Val Val 85 90 95 Arg Ser Cys Gly Cys His 100 (2)INFORMATION FOR SEQ ID NO: 160: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 102 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 160: Cys Arg Lys His Glu Leu Tyr Val Ser Phe GlnAsp Leu Gly Trp Gln 1 5 10 15 Asp Trp Ile Ile Ala Pro Lys Gly Tyr AlaAla Asn Tyr Cys Asp Gly 20 25 30 Glu Cys Ser Phe Pro Leu Asn Ala His MetAsn Ala Thr Asn His Ala 35 40 45 Ile Val Gln Thr Leu Val His Leu Met AsnPro Glu Tyr Val Pro Lys 50 55 60 Pro Cys Cys Ala Pro Thr Lys Leu Asn AlaIle Ser Val Leu Tyr Phe 65 70 75 80 Asp Asp Asn Ser Asn Val Ile Leu LysLys Tyr Arg Asn Met Val Val 85 90 95 Arg Ala Cys Gly Cys His 100 (2)INFORMATION FOR SEQ ID NO: 161: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 102 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 161: Cys Lys Lys His Glu Leu Tyr Val Ser Phe ArgAsp Leu Gly Trp Gln 1 5 10 15 Asp Trp Ile Ile Ala Pro Glu Gly Tyr AlaAla Tyr Tyr Cys Glu Gly 20 25 30 Glu Cys Ala Phe Pro Leu Asn Ser Tyr MetAsn Ala Thr Asn His Ala 35 40 45 Ile Val Gln Thr Leu Val His Phe Ile AsnPro Glu Thr Val Pro Lys 50 55 60 Pro Cys Cys Ala Pro Thr Gln Leu Asn AlaIle Ser Val Leu Tyr Phe 65 70 75 80 Asp Asp Ser Ser Asn Val Ile Leu LysLys Tyr Arg Asn Met Val Val 85 90 95 Arg Ala Cys Gly Cys His 100 (2)INFORMATION FOR SEQ ID NO: 162: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 102 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 162: Cys Arg Arg His Glu Leu Tyr Val Ser Phe GlnAsp Leu Gly Trp Leu 1 5 10 15 Asp Trp Val Ile Ala Pro Gln Gly Tyr SerAla Tyr Tyr Cys Glu Gly 20 25 30 Glu Cys Ser Phe Pro Leu Asp Ser Cys MetAsn Ala Thr Asn His Ala 35 40 45 Ile Leu Gln Ser Leu Val His Leu Met LysPro Asn Ala Val Pro Lys 50 55 60 Ala Cys Cys Ala Pro Thr Lys Leu Ser AlaThr Ser Val Leu Tyr Tyr 65 70 75 80 Asp Ser Ser Asn Asn Val Ile Leu ArgLys His Arg Asn Met Val Val 85 90 95 Lys Ala Cys Gly Cys His 100 (2)INFORMATION FOR SEQ ID NO: 163: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 102 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 163: Cys Gln Met Gln Thr Leu Tyr Ile Asp Phe LysAsp Leu Gly Trp His 1 5 10 15 Asp Trp Ile Ile Ala Pro Glu Gly Tyr GlyAla Phe Tyr Cys Ser Gly 20 25 30 Glu Cys Asn Phe Pro Leu Asn Ala His MetAsn Ala Thr Asn His Ala 35 40 45 Ile Val Gln Thr Leu Val His Leu Leu GluPro Lys Lys Val Pro Lys 50 55 60 Pro Cys Cys Ala Pro Thr Arg Leu Gly AlaLeu Pro Val Leu Tyr His 65 70 75 80 Leu Asn Asp Glu Asn Val Asn Leu LysLys Tyr Arg Asn Met Ile Val 85 90 95 Lys Ser Cys Gly Cys His 100 (2)INFORMATION FOR SEQ ID NO: 164: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 103 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 164: Cys Ala Arg Arg Tyr Leu Lys Val Asp Phe AlaAsp Ile Gly Trp Ser 1 5 10 15 Glu Trp Ile Ile Ser Pro Lys Ser Phe AspAla Tyr Tyr Cys Ser Gly 20 25 30 Ala Cys Gln Phe Pro Met Pro Lys Ser LeuLys Pro Ser Asn His Ala 35 40 45 Thr Ile Gln Ser Ile Val Arg Ala Val GlyVal Val Pro Gly Ile Pro 50 55 60 Glu Pro Cys Cys Val Pro Glu Lys Met SerSer Leu Ser Ile Leu Phe 65 70 75 80 Phe Asp Glu Asn Lys Asn Val Val LeuLys Val Tyr Pro Asn Met Thr 85 90 95 Val Glu Ser Cys Ala Cys Arg 100 (2)INFORMATION FOR SEQ ID NO: 165: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 102 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 165: Cys Lys Lys Arg His Leu Tyr Val Glu Phe LysAsp Val Gly Trp Gln 1 5 10 15 Asn Trp Val Ile Ala Pro Gln Gly Tyr MetAla Asn Tyr Cys Tyr Gly 20 25 30 Glu Cys Pro Tyr Pro Leu Thr Glu Ile LeuAsn Gly Ser Asn His Ala 35 40 45 Ile Leu Gln Thr Leu Val His Ser Ile GluPro Glu Asp Ile Pro Leu 50 55 60 Pro Cys Cys Val Pro Thr Lys Met Ser ProIle Ser Met Leu Phe Tyr 65 70 75 80 Asp Asn Asn Asp Asn Val Val Leu ArgHis Tyr Glu Asn Met Ala Val 85 90 95 Asp Glu Cys Gly Cys Arg 100 (2)INFORMATION FOR SEQ ID NO: 166: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 106 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 166: Cys Arg Ala Arg Arg Leu Tyr Val Ser Phe ArgGlu Val Gly Trp His 1 5 10 15 Arg Trp Val Ile Ala Pro Arg Gly Phe LeuAla Asn Tyr Cys Gln Gly 20 25 30 Gln Cys Ala Leu Pro Val Ala Leu Ser GlySer Gly Gly Pro Pro Ala 35 40 45 Leu Asn His Ala Val Leu Arg Ala Leu MetHis Ala Ala Ala Pro Gly 50 55 60 Ala Ala Asp Leu Pro Cys Cys Val Pro AlaArg Leu Ser Pro Ile Ser 65 70 75 80 Val Leu Phe Phe Asp Asn Ser Asp AsnVal Val Leu Arg Gln Tyr Glu 85 90 95 Asp Met Val Val Asp Glu Cys Gly CysArg 100 105 (2) INFORMATION FOR SEQ ID NO: 167: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 101 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 167: Cys His Arg His Gln Leu PheIle Asn Phe Gln Asp Leu Gly Trp His 1 5 10 15 Lys Trp Val Ile Ala ProLys Gly Phe Met Ala Asn Tyr Cys His Gly 20 25 30 Glu Cys Pro Phe Ser MetThr Thr Tyr Leu Asn Ser Ser Asn Tyr Ala 35 40 45 Phe Met Gln Ala Leu MetHis Met Ala Asp Pro Lys Val Pro Lys Ala 50 55 60 Val Cys Val Pro Thr LysLeu Ser Pro Ile Ser Met Leu Tyr Gln Asp 65 70 75 80 Ser Asp Lys Asn ValIle Leu Arg His Tyr Glu Asp Met Val Val Asp 85 90 95 Glu Cys Gly Cys Gly100 (2) INFORMATION FOR SEQ ID NO: 168: (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 103 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 168: Cys Arg Arg Thr Ser Leu His Val Asn Phe LysGlu Ile Gly Trp Asp 1 5 10 15 Ser Trp Ile Ile Ala Pro Lys Asp Tyr GluAla Phe Glu Cys Lys Gly 20 25 30 Gly Cys Phe Phe Pro Leu Thr Asp Asn ValThr Pro Thr Lys His Ala 35 40 45 Ile Val Gln Thr Leu Val His Leu Gln AsnPro Lys Lys Ala Ser Lys 50 55 60 Ala Cys Cys Val Pro Thr Lys Leu Asp AlaIle Ser Ile Leu Tyr Lys 65 70 75 80 Asp Asp Ala Gly Val Pro Thr Leu IleTyr Asn Tyr Glu Gly Met Lys 85 90 95 Val Ala Glu Cys Gly Cys Arg 100 (2)INFORMATION FOR SEQ ID NO: 169: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 105 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 169: Cys His Arg Val Ala Leu Asn Ile Ser Phe GlnGlu Leu Gly Trp Glu 1 5 10 15 Arg Trp Ile Val Tyr Pro Pro Ser Phe IlePhe His Tyr Cys His Gly 20 25 30 Gly Cys Gly Leu His Ile Pro Pro Asn LeuSer Leu Pro Val Pro Gly 35 40 45 Ala Pro Pro Thr Pro Ala Gln Pro Tyr SerLeu Leu Pro Gly Ala Gln 50 55 60 Pro Cys Cys Ala Ala Leu Pro Gly Thr MetArg Pro Leu His Val Arg 65 70 75 80 Thr Thr Ser Asp Gly Gly Tyr Ser PheLys Tyr Glu Thr Val Pro Asn 85 90 95 Leu Leu Thr Gln His Cys Ala Cys Ile100 105 (2) INFORMATION FOR SEQ ID NO: 170: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 99 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 170: Cys Ala Leu Arg Glu Leu SerVal Asp Leu Arg Ala Glu Arg Ser Val 1 5 10 15 Leu Ile Pro Glu Thr TyrGln Ala Asn Asn Cys Gln Gly Ala Cys Gly 20 25 30 Trp Pro Gln Ser Asp ArgAsn Pro Arg Tyr Gly Asn His Val Val Leu 35 40 45 Leu Leu Lys Met Gln AlaArg Gly Ala Thr Leu Ala Arg Pro Pro Cys 50 55 60 Cys Val Pro Thr Ala TyrThr Gly Lys Leu Leu Ile Ser Leu Ser Glu 65 70 75 80 Glu Arg Ile Ser AlaHis His Val Pro Asn Met Val Ala Thr Glu Cys 85 90 95 Gly Cys Arg (2)INFORMATION FOR SEQ ID NO: 171: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 102 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 171: Cys Glu Leu His Asp Phe Ser Leu Ser Phe SerGln Leu Lys Trp Asp 1 5 10 15 Asn Trp Ile Val Ala Pro His Ser Tyr AsnPro Ser Tyr Cys Lys Gly 20 25 30 Asp Cys Pro Ser Ala Val Ser His Arg TyrGly Ser Pro Val His Thr 35 40 45 Met Val Gln Asn Met Ile Tyr Glu Lys LeuAsp Pro Ser Val Pro Ser 50 55 60 Pro Ser Cys Val Pro Gly Lys Tyr Ser ProLeu Ser Val Leu Thr Ile 65 70 75 80 Glu Pro Asp Gly Ser Ile Ala Tyr LysGlu Tyr Glu Asp Met Met Ala 85 90 95 Thr Ser Cys Thr Cys Arg 100 (2)INFORMATION FOR SEQ ID NO: 172: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 94 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 172: Cys Val Leu Thr Ala Ile His Leu Asn Val Thr Asp Leu GlyLeu Gly 1 5 10 15 Tyr Glu Thr Lys Glu Glu Leu Ile Phe Arg Tyr Cys SerGly Ser Cys 20 25 30 Asp Ala Ala Glu Thr Thr Tyr Asp Lys Ile Leu Lys AsnLeu Ser Arg 35 40 45 Asn Arg Arg Leu Val Ser Asp Lys Val Gly Gln Ala CysCys Arg Pro 50 55 60 Ile Ala Phe Asp Asp Asp Leu Ser Phe Leu Asp Asp AsnLeu Val Tyr 65 70 75 80 His Ile Leu Arg Lys His Ser Ala Lys Arg Cys GlyCys Ile 85 90 (2) INFORMATION FOR SEQ ID NO: 173: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 95 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 173: Cys Gly Leu Arg Glu Leu GluVal Arg Val Ser Glu Leu Gly Leu Gly 1 5 10 15 Tyr Ala Ser Asp Glu ThrVal Leu Phe Arg Tyr Cys Ala Gly Ala Cys 20 25 30 Glu Ala Ala Ala Arg ValTyr Asp Leu Gly Leu Arg Arg Leu Arg Gln 35 40 45 Arg Arg Arg Leu Arg ArgGlu Arg Val Arg Ala Gln Pro Cys Cys Arg 50 55 60 Pro Thr Ala Tyr Glu AspGlu Val Ser Phe Leu Asp Ala His Ser Arg 65 70 75 80 Tyr His Thr Val HisGlu Leu Ser Ala Arg Glu Cys Ala Cys Val 85 90 95 (2) INFORMATION FOR SEQID NO: 174: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 291 base pairs (B)TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 174:GCCTTGGCTG GTTCATGCCG ACTGTGGAGC CTGACCCTAC CAGTGGCTGA GCTGGGCCTG 60GGCTATGCCT CGGAGGAGAA GGTCATCTTC CGATACTGTG CTGGCAGCTG TCCCCAAGAG 120GCCCGTACCC AGCACAGTCT GGTACTGGCC CGGCTTCGAG GGCGGGGTCG AGCCCATGGC 180CGACCCTGCT GCCAGCCCAC CAGCTATGCT GATGTGACCT TCCTTGATGA TCAGCACCAT 240TGGCAGCAGC TGCCTCAGCT CTCAGCTGCA GCTTGTGGCT GTGGTGGCTG A 291 (2)INFORMATION FOR SEQ ID NO: 175: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 405 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 175: GTAAGAATTC CTGGGGGCCT CCCGACTCCC CAATTCCTTCTCTCAAAGCC CTCACTTTGC 60 CTTACAATCC TACTCTACCT TGCACTAGGT AACAACCATGTCCGTCTTCC AAGAGCCTTG 120 GCTGGTTCAT GCCGACTGTG GAGCCTGACC CTACCAGTGGCTGAGCTGGG CCTGGGCTAT 180 GCCTCGGAGG AGAAGGTCAT CTTCCGATAC TGTGCTGGCAGCTGTCCCCA AGAGGCCCGT 240 ACCCAGCACA GTCTGGTACT GGCCCGGCTT CGAGGGCGGGGTCGAGCCCA TGGCCGACCC 300 TGCTGCCAGC CCACCAGCTA TGCTGATGTG ACCTTCCTTGATGATCAGCA CCATTGGCAG 360 CAGCTGCCTC AGCTCTCAGC TGCAGCTTGT GGCTGTGGTGGCTGA 405 (2) INFORMATION FOR SEQ ID NO: 176: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 291 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA(genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 176: GCCTTACCTGGTTTGTGCCG GCTGTGGAGC CTGACCCTAC CAGTGGCTGA GCTTGGCCTG 60 GGCTATGCCTCAGAGGAGAA GATTATCTTC CGATACTGTG CTGGCAGCTG TCCCCAAGAG 120 GTCCGTACCCAGCACAGTCT GGTGCTGGCC CGTCTTCGAG GGCAGGGTCG AGCTCATGGC 180 AGACCTTGCTGCCAGCCCAC CAGCTATGCT GATGTGACCT TCCTTGATGA CCACCACCAT 240 TGGCAGCAGCTGCCTCAGCT CTCAGCCGCA GCTTGTGGCT GTGGTGGCTG A 291

What is claimed is:
 1. A method for promoting the survival of amesencephalic neuronal cell comprising administering to the cell apersephin polypeptide; said polypeptide comprising an amino acidsequence that contains amino acids at positions indicated inparentheses: Cys (1), Leu (3), Val (10), Leu (13), Gly (14), Leu (15),Gly (16), Tyr (17), Glu (21), Phe (25), Arg (26), Tyr (27), Cys (28),Gly (30), Cys (32), Leu (44), Leu (47), Cys (58), Cys (59), Pro (61),Asp (66), Phe (69), Leu (70), Asp (71), Ser (83), Ala (84), Cys (87),and Cys (89), said positions being identified by alignment with a mousepersephin sequence as set forth in SEQ ID NO:79 or a rat persephinsequence as set forth in SEQ ID NO:82; wherein said persephin amino acidsequence has at least 96% sequence identity with SEQ ID NO:79 or SEQ IDNO:82 and wherein said persephin polypeptide promotes survival ofmesencephalic neuronal cells.
 2. The method of claim 1, wherein thepersephin polypeptide is produced by recombinant methods.
 3. The methodof claim 1, wherein the persephin polypeptide is SEQ ID NO:80.
 4. Themethod of claim 1 wherein the persephin polypeptide comprises SEQ IDNO:79 or SEQ ID NO:82.
 5. The method of claim 1 wherein the persephinpolypeptide consists of SEQ ID NO:79 or SEQ ID NO:82.